【作者】 范雁；

【导师】 黄瑞； 朱梦漾；

【作者基本信息】 苏州大学 ， 免疫学， 2010， 博士

【摘要】 Phox2a和Phox2b是两个同源异型结构域蛋白,在胚胎形成过程中控制去甲肾上腺素能神经元的分化。在中枢神经系统,它们在所有的去甲肾上腺素能神经元中表达,而且是去甲肾上腺素能表型的决定因素。作为去甲肾上腺素能循环的标志蛋白,转录因子Phox2a和Phox2b对多巴胺β-羟化酶(dopamineβ-hydroxylase,DBH)和去甲肾上腺素转运体(norepinephrine Transporter,NET)表达的调节和蓝斑(locus coeruleus,LC)神经元的发生与解离紧密联系。近年来,病毒载体已经成功地用于介导基因或小分子干扰RNA进入哺乳动物神经元或脑。然而,这个途径的缺点是病毒载体的表达会随时间而消退,表达的高峰出现在2-3天,7天后就消失了。如何将基因高效转染及长期稳定表达成为人们研究的热点。近期研究者把目光投向了以Ⅰ型人免疫缺陷病毒(human immunodeficiency virus type 1,HIV1)为代表的慢病毒,不仅具有可感染非分裂期的细胞、目的基因整合至靶细胞基因组以及长期表达、免疫反应小等优点,而且对重组包膜质粒、包装质粒及重组目的基因穿梭质粒进行改造后,极大降低了其自我复制能力,使安全性大大提高,有望成为理想的基因载体。本研究采用RT-PCR方法获得大鼠Phox2a和Phox2b全外显子片段,将其克隆入慢病毒表达载体并进行序列测定,利用293FT包装细胞制备了Phox2a/2b与增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)融合基因慢病毒。注射病毒浓缩液至大鼠LC部位,介导Phox2a和Phox2b在LC长期稳定表达,为研究Phox2a和Phox2b在去甲肾上腺素能系统中的作用及机制提供适合的稳定转染细胞的载体,为基因治疗一些神经退化性疾病提供理论和实验依据。本实验分两部分:一、用携带人类Phox2a/2b cDNA的pCMV6-XL5载体以及对人类Phox2a/2b基因特异的shRNA分别转染SK-N-BE(2)C细胞,从过表达、封闭基因表达两方面探索Phox2基因对分化成熟的去甲肾上腺素能神经元细胞中NET和DBH表达的影响。二、克隆大鼠Phox2基因并构建能稳定表达Phox2的慢病毒载体,通过慢病毒包装体系生成能有效感染分裂细胞和非分裂细胞的重组慢病毒颗粒;注射此重组慢病毒至大鼠LC部位,一段时间后观察Phox2基因的过表达对成年大鼠脑组织相关部位NET和DBH表达的影响。本研究首次探索转录因子(包括Phox2a/2b)和成熟的去甲肾上腺素能神经系统之间可能的关系,为深入了解帕金森氏病(Parkinson’s disease ,PD)、老年痴呆症(Alzheimer’s disease,AD)及重症抑郁(Major depression)等神经性疾病的发病机制提供了重要的理论和实验依据;构建的高效持久的携带Phox2基因的慢病毒表达载体为这些神经性疾病的临床治疗提供新的途径。第一部分转录因子phox2对SK-N-BE(2)C细胞中去甲肾上腺素传递体(NET)和多巴胺β-羟化酶(DBH)表达的影响目的:Phox2a和Phox2b是两个同源异型结构域蛋白,在胚胎形成过程中控制去甲肾上腺素能神经元的分化。NET和DBH是去甲肾上腺素能系统的两个重要标志物。本研究检测了Phox2a/2b在体外对NET和DBH表达的影响,从而了解Phox2基因对分化的去甲肾上腺素能神经元细胞的调控作用。方法:1.培养SK-N-BE(2)C细胞至90%汇合度时,分别用0.1、1、5μg携带人类Phox2a/2b cDNAs的pCMV6-XL5质粒,由脂质体Lipofectamine 2000介导转染细胞,对照组则转染pCMV6-XL5空载体。转染后3~4天收集各组细胞,RT-PCR法检测Phox2a或Phox2b、NET、DBH、TH等在mRNA水平的表达;Western Blot检测各指标在蛋白水平的表达;NET的转运功能以再摄取[3H]标记的去甲肾上腺素能力来判断。2. SK-N-BE(2)C细胞培养至50~60%汇合度时,用Arrest-In转染试剂将2μg对Phox2a或Phox2b特异的shRNA质粒DNA转染细胞,对照组按相同步骤转染pLKO.1空载体。转染5~7天后收集各组细胞,按与1相同的方法检测各指标。3.为证明Phox2a或Phox2b是否单独地调控甲肾上腺素能表型,将2μg对Phox2a(或Phox2b)特异的shRNA质粒与5μg Phox2b(或Phox2a)cDNA同时转染SK-N-BE(2)C细胞,对照组则同时转染pCMV6-XL5和pLKO.1空载体,3~4天后收集各组细胞测定DBH在mRNA、蛋白质水平的表达变化。结果:0.1~5μg的Phox2a/2b cDNAs能明显提高NET和DBH在mRNA及蛋白质水平的表达,且呈剂量相关性。转染后NET表达的增加相应地表现出[3H]标记的去甲肾上腺素摄入的增加。和单独转染Phox2a或Phox2b相比,共转染Phox2a和Phox2b并没有表现出对NET和DBH表达的协同促进作用。转染对Phox2a或Phox2b基因特异的shRNA后,通过关闭内源性Phox2a/2b,显著降低NET和DBH在mRNA及蛋白质水平的表达,同时伴随着[3H]标记的去甲肾上腺素摄取的下降。而且,共转染Phox2a和Phox2b基因特异的shRNAs后,其对NET的mRNA水平表达的影响具有累加效应。最后,转染Phox2a基因特异的shRNA所引起的DBH表达的下降可以通过转染Phox2b基因的cDNA来逆转,反之亦然。证明Phox2a和Phox2b对SK-N-BE(2)C细胞中DBH表达水平影响的互换效应。结论:该研究在体外证实了Phox2a和Phox2b对NET和DBH表达及生物学功能的调控作用,深入地阐明Phox2a/2b这两个转录因子对去甲肾上腺素能系统中关键蛋白的调节作用,可能开辟了治疗老年引起的去甲肾上腺素能系统功能失调的新途径。第二部分携带EGFP和大鼠phox2融合基因重组慢病毒载体的构建及其对大鼠脑去甲肾上腺素能神经元的调控作用目的:克隆全长大鼠转录因子Phox2 cDNA,制备大鼠Phox2基因与增强型绿色荧光蛋白(EGFP)融合基因慢病毒,病毒体外感染293FT细胞和体内感染大鼠LC部位,以确定病毒携带的Phox2基因表达效率,探索Phox2基因的神经生物学功能。方法:采用RT-PCR方法获得大鼠Phox2a/2b全外显子片段,运用TOPO克隆技术使之克隆到慢病毒表达载体质粒pLenti6/V5-DEST中。经限制性酶切、PCR扩增和测序鉴定重组载体。在脂质体介导下将慢病毒包装系统的包装结构基因pCMV△8.9、包膜基因VSVG和目的基因pLenti6/V5-EGFP-Phox2a/2b导入病毒包装细胞293FT,荧光显微镜检测基因的表达。包装成病毒后,收集病毒上清,浓缩。未浓缩的病毒液稀释到不同浓度梯度转染HT1080细胞,杀稻瘟素(Blasticidine)筛选抗性细胞克隆,计数细胞克隆后确定病毒滴度。制备的病毒感染293FT细胞,3天后荧光显微镜检测基因的表达,收集细胞以Western Blot法检测Phox2基因在蛋白质水平的表达。将大鼠分4组:对照组、假手术组、注射重组Phox2a病毒组和Phox2b组。浓缩的病毒(2~3μl,1x108 TU/ml)立体定位微量注射至大鼠LC部位,不同时间段(7 d,14 d,21 d)取大鼠蓝斑(LC)、海马(hippocampus ,HP)和嗅球(olfactory bulb ,OB)等组织,通过原位杂交(In Situ Hybridization)、Western Blot、免疫组化(Immunohistochemistry,IHC)、BrdU标记等方法检测各组Phox2、NET、DBH、TH及神经元细胞增殖(Neurogenesis)等的变化。结果:限制性内切酶酶切和DNA测序分析证实RT-PCR获得的大鼠Phox2a/2b cDNA片段与GenBank中公布的数据基本吻合; EGFP与大鼠Phox2a/2b融合基因准确克隆入pLenti6/V5-DEST的多克隆位点。慢病毒的3种质粒可高效转染入293FT细胞,荧光显微镜下观察可见大量的绿色荧光。未浓缩病毒液的滴度可达5×106 TU/ml。病毒感染293FT细胞后Phox2基因在蛋白质水平的表达有明显的增加。原位杂交结果显示,注射重组Phox2a/2b病毒组在不同时间点,Phox2a和Phox2b在LC部位的mRNA水平均有显著升高,注射后14天Phox2a增加最多,达146%;注射后7天Phox2b增加最多,达41%。与对照组及假手术组相比,Phox2a和Phox2b的过表达分别伴随着NET和DBH mRNA水平的明显增加,NET分别增加了78.2%和110%,DBH分别增加了42.2%和39.3%;同样,在HP部位,Western Blot结果显示,和对照组及假手术组相比,Phox2a和Phox2b的过表达也分别伴随着NET和DBH在蛋白质水平的明显增加,NET分别增加了78.4%和48.6%,DBH分别增加了37.6%和32.3%;在OB部位也获得相似的实验结果;BrdU标记结果证明由Phox2a/2b导致的NET、DBH表达的增加可以促进LC部位神经元细胞的增殖,尤其是联合注射重组Phox2a和Phox2b病毒组对神经元细胞增殖的效应最明显。结论:成功制备了大鼠Phox2与EGFP融合基因慢病毒,为研究Phox2基因在去甲肾上腺素能系统的发生分化等过程中的作用机制提供合适的稳定转染细胞的载体;体内外试验证实上调Phox2的表达可促进成熟大鼠脑组织中NET和DBH的表达,预示了Phox2基因对维持大鼠出生后去甲肾上腺素能神经元的功能发挥着重要作用。更多还原

【Abstract】 Part I Effects of Transcription Factors Phox2 on Expression of Norepinephrine Transporter and Dopamineβ–hydroxylase in SK-N-BE(2)C cellsObjective: Phox2a and Phox2b are two homeodomain proteins that control the differentiation of noradrenergic neurons during embryogenesis. Norepinephrine transporter (NET) and dopamineβ-hydroxylase (DBH) are two important markers of the noradrenergic system. In the present study, we examined the possible effect of Phox2a/2b on the in vitro expression of NET and DBH to understand the regulatory function of Phox2a/2b on differentiated noradrenergic neuron cells in vitro.Methods: (1) The human neuroblastoma cell line SK-N-BE(2)C cells were grown to about 90% confluence on 6-well plates and transfected with 0.1, 1, 5μg pCMV6-XL5 plasmid DNA carrying cDNAs of human Phox2a or Phox2b, using Lipofectamine 2000 reagent. For the control group, cells were transfected with pCMV6-XL5 empty vectors. Cells were harvested 3~4 days after transfection for different measurements. The mRNA and protein level of Phox2a or Phox2b, NET, DBH and TH were measured by RT-PCR or Western Blot. The transport ability of NET was detected by uptake of [3H]NE. (2) When confluence of cultured SK-N-BE(2)C cells arrived to 50~60% on 6-well plates, 2μg shRNAs specific to Phox2a or Phox2b genes were transfected into cells mediated by Arrest-In reagent kits. Cells in the control group were transfected with pLKO.1 empty vectors based on the instruction from the manufacturer by the same procedure. Cells were harvested 5~7 days after transfection for different measurements using the same methods mentioned above. (3) In order to examine whether either Phox2a or Phox2b independently regulated the expression of the noradrenergic phenotype, two similar experiments were carried out. In the first experiment, SK-N-BE(2)C cells were transfected with either 2μg shRNA specific to Phox2a or this shRNA plus 5μg Phox2b cDNA for 5 (for RT-PCR) or 7 days (for western blotting). Another experiment was carried out with Phox2b shRNA and Phox2a cDNA using the same parameters as the first one. The mRNA and protein levels of DBH were measured.Results: Transfection of 0.1 to 5μg of cDNAs of Phox2a or Phox2b significantly increased mRNA and protein levels of NET and DBH in a concentration-dependent manner. As a consequence of the enhanced expression of NET after transfection, there was a parallel increase in the uptake of [3H] norepinephrine. Co-transfection of Phox2a and Phox2b did not further increase the expression of noradrenergic markers when compared with transfection of either Phox2a or Phox2b alone. Transfection of shRNAs specific to Phox2a or Phox2b genes significantly reduced mRNA and protein levels of NET and DBH after shutdown of endogenous Phox2, which was accompanied by a decreased [3H] norepinephrine uptake. Furthermore, there was an additive effect after cotransfection with both shRNAs specific to Phox2a or Phox2b genes on NET mRNA levels. Finally, the reduced DBH expression caused by the shRNA specific to Phox2a could be reversed by transfection with Phox2b cDNA and vice versa, which also indicates that in SK-N-BE(2)C cells there is a functionally reciprocal effect of Phox2a and Phox2b on DBH expression.Conclusions: The present findings verify the determinant role of Phox2a and Phox2b on the expression and function of NET and DBH in vitro. Further clarifying the regulatory role of these two transcription factors on key proteins of the noradrenergic system may open a new avenue for therapeutics of aging-caused dysfunction of the noradrenergic system.Part II Construction of Recombinant Lentiviral-vector with EGFP-Phox2 Fusion Genes and Their Regulatory Effects on Noradrenergic Neuron System in Rat BrainsObjective: Lentivirus mediated gene expression is one of the most promising methods to produce transgenic animals. To prepare high titer of lentiviral particle packaging is the key of this technology. Phox2a and Phox2b, two homeodomain transcriptional factors, are determinants of the noradrenergic phenotype during embryogenesis. However, their roles for the function of the noradrenergic neurons in mature brains remain unknown. The norepinephrine transporter (NET) and dopamineβ-hydroxylase (DBH) are two important markers of the noradrenergic system. In the present study, possible effects of Phox2a/2b on expression of the NET and DBH in the brain were examined. First, cDNAs of rat Phox2a and Phox2b were cloned. Then the recombinants of pLenti6/V5-DEST vector carrying EGFP-Phox2a/2b fusion genes were constructed. These lentiviral-gene constructs were further transducted into 293FT cells to test the expression level of Phox2 genes. Finally, lentiviral vectors carrying cDNAs of Phox2a/2b were microinjected into the locus coeruleus (LC) in adult Fischer 344 rats.Methods: The cDNA sequences of rat Phox2a and Phox2b were cloned by RT-PCR. Using pENTRTM Directional TOPO cloning technology, EGFP-Phox2 fusion genes were inserted into pLenti6/V5-DEST expression vectors to construct two recombinant entry vectors. These two recombinants were analyzed by restriction analysis, PCR reaction, and sequencing to confirm the presence and correct orientation of the insert. Then, 293FT cells were cotransfected with three plasmids: pCMVΔR8.9, pVSVG and pLenti6/V5-EGFP-phox2a/2b, using the TransIT?-293 Transfection Reagent kit. After observed strong fluorescences under fluorencent microscope, virus supernatant was collected after 48-72 hours, and concentrated by ultracentrifuge. Two further experiments were performed. 1) Testing the titer of virus preparation: HT1080 cells were transduced with 10 fold serial dilutions ranging from 10-2~10-6 of lentivirus and the positive transduced cells were selected with 4mg/ml Blasticidine. The titer was determined by counting the blue-stained colonies. 2) Testing the expression of Phox2a/2b: 293FT cells was transduced using different concentration of lentiviral preparation stock for three days. After observed the fluorescence, cells were harvested to examine Phox2a/2b protein level by Western Blot. After all these pretests verified the viral titer and expressional ability of Phox2a/2b in vitro, the stereotaxic microinjection experiments were started. The concentrated lentivirus stock (2~3μl, ~108TU/ml) was microinjected into the LC region of rats. These rats were sacrificed after 7, 14, 21 days of injection. The brain LC, hippocampus and frontal cortex were sectioned or dissected for different measurements. mRNA levels of Phox2, NET and DBH in the LC were determined by in situ hybridization. Protein levels of NET and DBH in the hippocampus and frontal cortex were measured by Western Blot. The neurogenesis in the hippocampus was determined by BrdU immunostaining.Results: The full length of rat Phox2 cDNA was successfully cloned. The constructs of recombinant expression vector pLenti6/V5-EGFP-Phox2a/2b were confirmed by restriction analysis, PCR reaction, and sequencing. After 293FT cells were effectively cotransfected with these constructs, a lentiviral stock was produced and a satisfactory EGFP expression was verified by fluorescence microscopic analysis. The nonconcentrated virus titer reached higher than 5x106 TU/ml. In vitro expressional tests proved that transfection with Lentiviral vectors carrying cDNAs of Phox2a/2b increased mRNA and protein levels of Phox2 genes, indicating that a high titer lentiviral packaging platform was preliminary established. In situ hybridization showed that microinjection with cDNAs of Phox2a and Phox2b for different time periods significantly increased their mRNAs in the LC regions with highest levels on 14 days (for Phox2a, increased by 146%) and 7 days (for Phox2b, increased by 41%) post-injection, respectively. Compared to those in the control and sham operation groups, overexpressions of Phox2a and Phox2b were respectively paralleled by a significant increase in NET mRNA levels by 78.2% and 110%; and a significantly increased DBH mRNA level by 42.2% and 39.3%. Similarly, microinjection with cDNAs of Phox2a and Phox2b markedly increased protein levels of NET in the hippocampus by 78.4% and 48.6%; and those of DBH by 37.6% and 32.3%, respectively. BrdU immunostaining results showed that overexpression of Phox2a/2b in the LC stimulated the neurogenesis in the hippocampus area, especially after the injection with lentivirus carrying both Phox2a and Phox2b cDNAs.Conclusion: The present study presented a successful construction of two recombinant lentivirus vectors which contained genes of rat Phox2a and Phox2b respectively. With these constructs, the present study demonstrates an upregulatory effect of Phox2a and Phox2b on the expression of NET and DBH in the mature rat brains, indicating that Phox2 genes may play an important role in maintaining the function of the noradrenergic neurons after birth. These results may indicate the possible therapeutic strategy for the treatment of noradrenergic deficiency occurred in the neuronal degeneration diseases. Also, these methodologies will be benefit of the further study to produce transgenic animals.更多还原