摘要
【背景】提高损伤后视网膜神经节细胞(RGCs)存活和再生能力的研究是眼科和神经生物学科共同关注的热点和焦点。具有神经保护性效能的神经营养因子是实现视觉系统中枢神经修复基因治疗和移植的前提和物质基础。然而,既往的研究多集中在某种生长和营养因子的单一研究,迄今为止,还未能找到一种因子能对RGC提供持续的营养支持作用足以克服其周围的再生抑制环境而促进损伤的轴突再生。如果能寻找到某种在转录水平或转录前作用的因子,则该因子有可能启动RGC生长的极链式反应,轴突再生的难题也就迎刃而解了。晶状体来源的上皮生长因子(lens epithelium derived growth factor, LEDGF)是2000年新发现的一种新的生长、粘附、分化、抗凋亡和存活因子,它能提高多数细胞生长粘附能力、促进其分化、延长其存活。LEDGF基因位于人类染色体9p21,该基因选择性的剪切而产生两种蛋白,即LEDGFp75和LEDGFp52蛋白。相比LEDGFp52而言,LEDGFp75只是一个弱的转录辅活化子。最近的研究表明LEDGFp75能促进多种细胞的生长与存活,譬如对晶状体上皮细胞、视网膜光感受器细胞、视网膜色素上皮细胞所发挥的生长刺激和促进存活效应。进一步的研究发现LEDGF在脑内的发育及区域表达,提示其参与神经上皮干细胞分化及神经发生。因此,有专家提议将LEDGF作为视网膜疾病的治疗性蛋白和神经营养因子转染的候选分子。基于LEDGFp75具有一定的眼部保护作用的研究前提、联系LEDGFp52可能比LEDGp75生物学作用更强的生物信息学预测、结合国际上对LEDGFp52这一转录活化子研究很少的事实和视神经再生研究中积极寻找具有神经保护和生长促进效能神经营养因子的要求,我们选择LEDGFp52作为研究的目标。那么,LEDGFp52作为一个新发现的因子,它是否能对RGC起到神经保护和生长促进作用呢?它对RGC神经元生长相关基因和蛋白又有什么影响呢?这有待于我们通过实验研究来明确。
【目的】在原代大鼠RGCs培养的基础上进行LEDGFp52基因和蛋白两个水平正负双向调控实验,观察它们对大鼠RGCs突起数目及轴突长度、RGC神经元特异性相关基因和蛋白的调控,全面了解LEDGFp52基因和蛋白对大鼠RGCs生长的影响,为研发更有效的RGC生长和营养因子奠定基础,为探索视神经损伤修复的新途径提供实验依据。
【方法】NEUROBASALTM Media无血清体系培养RGCs,采用RT-PCR和细胞免疫荧光化学检测LEDGFp52基因和蛋白在RGCs表达;构建rhLEDGFp52基因原核表达载体、诱导表达及纯化蛋白;细菌内同源重组法构建LEDGFp52基因腺病毒载体、体外表达及制备病毒;构建siRNA-LEDGFp52真核表达载体、鉴定该载体并检测其对LEDGFp52蛋白的抑制率。
1、LEDGFp52基因和蛋白对大鼠RGCs的突起数目及轴突长度调控作用的观察:分为实验组和对照组,实验组在大鼠RGCs培养36h后分别将LEDGFp52(2×10-4g/L)和Ab-LEDGF(2.5×10-4g/L)加入RGCs的无血清培养基中,在LipofectamineTM2000介导下将Ad-LEDGFp52(2.5×10-4g/L)(3×10-4pfu/L)和siRNA-LEDGFp52(6×10-4g/L)转染进RGC,分别在处理后12h、24h、36h、48h、72h和96h在相差显微镜下观察。设阳性对照组(CNTF10-4g/L)和空白对照组(不加处理因素),观察时相点同上。对照组及实验组的每组观察孔数为12孔,重复3次。检测指标:RGCs轴突长度和单个细胞的突起数目。分析软件:IPP图像分析系统。
2、LEDGFp52基因和蛋白对RGC神经元生长相关基因和蛋白调控效应的研究:处理因素及分组同上。检测指标:主要采用RT-PCR和细胞免疫荧光化学检测技术进一步研究LEDGFp52对RGC神经元生长相关的GAP-43、NF-L和MAP-2基因与蛋白的调控。分析软件:Quantity One程序半定量分析各个目的基因在mRNA水平的相对含量,以待测基因与相应内参照光密度比值计算相对系数;LSM510-Meta-Expert程序半定量分析各个目的蛋白的平均荧光强度相对数值。
【结果】
1、无血清的NEUROBASALTM Media培养基是一种较好的RGCs培养体系;LEDGFp52基因及其蛋白在RGCs均有表达。
2、利用基因重组技术将rhLEDGFp52基因构建于原核表达载体pET30a(+)中,经酶切、测序鉴定证实构建完全正确,通过IPTG诱导其在大肠杆菌BL21(DE3)中获得可溶性形式表达,rhLEDGFp52蛋白表达量占菌体蛋白总量的34.63%。Western Blot结果显示rhLEDGF2蛋白能够特异性与LEDGF-ab结合。Ni-NTA His.Bind.Resin方法进行纯化后的rhLEDGFp52,终浓度达520μg.mL-1,分析其纯度达87.93%。
3、成功将LEDGFp52基因片段亚克隆到腺病毒穿梭质粒pAdTrack-CMV上构建腺病毒穿梭载体pAdTrack-CMV-LEDGFp52,并将腺病毒穿梭载体pAdTrack-CMV-LEDGFp52与5型腺病毒骨架质粒pAdeasy-1共转染BJ5183细菌,经细菌内同源重组产生携带LEDGFp52基因重组腺病毒载体pAd-LEDGFp52,将pAd-LEDGFp52经脂质体法转化293细胞包装产生重组腺病毒Ad-LEDGFp52,滴度可达5×1012 pfu.L-1。将Ad-LEDGFp52体外转染293细胞,在该细胞中有效表达目的基因LEDGFp52,CPE法及Westernblot均证实了该目的基因表达。
4、成功构建LEDGFp52基因RNA干扰(RNAi)的真核细胞表达载体,重组质粒转染HeLa细胞48小时,Westernblotting检测到LEDGFp52蛋白表达的改变,Quantity One程序半定量分析LEDGFp52蛋白明显下调了70%。
5、LEDGFp52在基因水平和蛋白水平均可调控RGCs生长,表现为正向调节RGCs轴突显著增长,负向调节RGCs轴突显著缩短,而且正向调控时有高峰效应。
6、LEDGFp52是RGC树突化因子,同时也是RGC轴突延伸因子。
7、LEDGFp52在基因和蛋白两个水平对RGC神经元生长相关的基因/蛋白GAP-43、NF-L和MAP-2的表达均有显著的调控作用,表现为正向调节RGCs神经元生长相关基因/蛋白表达升高,负向调节RGCs神经元生长相关基因/蛋白表达降低。
【结论】
1.首次确立了LEDGFp52基因及其蛋白在RGCs的表达。
2.利用基因重组技术成功获得rhLEDGFp52蛋白,终浓度达520μg.mL-1,纯度达87.93%;经细菌内同源重组成功制备LEDGFp52的重组腺病毒,滴度可达5×1012 pfu.L-1,并能够在真核细胞中获得高效稳定的表达;利用RNAi技术可成功构建抑制LEDGFp52表达的小干扰RNA重组体,该重组体使LEDGFp52蛋白明显下调70%。
3. LEDGFp52基因和蛋白能显著调控大鼠RGCs单个细胞突起数目及轴突平均长度,其作用强于CNTF。
4. LEDGFp52是RGC树突化因子,也是RGC轴突延伸因子。
5. LEDGFp52基因和蛋白通过调控RGC神经元生长相关的基因/蛋白GAP-43、NF-L和MAP-2的表达促进RGC生长。
6. LEDGFp52基因在调控RGC树突化时有其它树突化因子的参与。
Backgrounds
The study on enhancing survival and regeneration of injured RGCs has been a common hot spot and focus in ophthalmological research as well as neurobiological investigation. Neuroprotective factor is a prerequisite and substance foundation of gene therapy and transplantation implement in CNS restoration of visual system. However, in the past a few years, the study on neuroprotective factor just focused on the factor itself, up to now, we haven’t attained a certain factor which could provide persistent nutritional support for injured RGCs to surmount their regeneration inhibited circumstance and promote axonal outgrowth. If we can find a special factor which could play an important role on transcription or pre-transcription level, perhaps the factor could initiate the growth-chain-reaction of RGCs, the tough problem on axonal regeneration will be solved. LEDGF is a novel growth, adhering, differentiation, anti-apoptosis and survival factor, which was isolated from the cDNA Lib in 2000. The gene of LEDGF was located in 9p21, and it can be alternative splicing produced two isoforms—LEDGFp52 and LEDGFp75. LEDGFp75 was just a lepto-transcription coactivator compared with LEDGFp52, and previous investigation has identified that LEDGFp75 had neuroprotective effect, it can elevate growth and adhering abilities of most cells, such as LEC, RPE and RPR of ocular cells, promote their differentiation and prolong their survival. Sequential researches revealed that LEDGF was regionally expressed in developing brain, and it was involved in the process of neuroepithelia stem cell differentiation and neurogenesis. Therefore, some specialists have suggested LEDGFp75 should be a candidate for remedial protein and neuronutritional transfection factor of retinal desease. Based on the research of neuroprotective effect of LEDGFp75 on some ocular cells, in accordance with the bioinformatics inference that LEDGFp52 is maybe much more competent than LEDGFp75, cohered to the reality that study on LEDGFp52 is very rare in the world, and coped with the requirement of neuroprotective and growth-promoting nutritional factor for RGCs axonal regeneration, we chose LEDGFp52 as our research target. Thus, to be a relatively novel revelation, does LEDGFp52 have a growth-promoting effect on RGCs? Can LEDGFp52 regulate the expression of RGCs neuronal specificity associated gene & protein? An empirical study is necessary to identifying these issues.
Purposes
To positively and negatively regulate LEDGFp52 both on gene level and protein level on the base of primary RGCs culturing; to observe their impact on the prominence numbers & axonal length of rat RGCs, to investigate their influence to RGC neuronal specificity associated gene & protein; to understand the effect of LEDGFp52 gene &protein on rat RGCs growth, to lay a foundation for seeking more competent growth and nutrition factor of RGC,to furnish exploration on new path of injured optic nerve recovery with experimental proof .
Methods
Neurobasal TM Media serum-free system was used in culturing RGCs, RT-PCR and CMIFC was applied to detect the expression of LEDGFp52 gene & protein on RGCs; rhLEDGFp52 prokaryotic expression vector was constructed, rhLEDGFp52 protein expression was induced and purified; LEDGFp52 adenovirus vector was constructed by homologous recombination in bacteria, in vitro expression was executed and virus was prepared; siRNA-LEDGFp52 eukaryotic expression vector was constructed and identified, and the inhibition ratio on LEDGFp52 protein was examinated.
1. Observation of LEDGFp52 gene & protein effect on prominence numbers & axonal length of rat RGCs: experimental groups and control groups were divided. In experiment groups, LEDGFp52(2×10-4g/L) and Ab-LEDGF(2.5×10-4g/L) were added into the RGCs serum-free media, Ad-LEDGFp52(2.5×10-4g/L)(3×10-4pfu/L) and siRNA-LEDGFp52(6×10-4g/L) were transfected into RGCs by Lipofectamine TM 2000 while RGCs culturing 36hours. In treatment 12h, 24h, 36h, 48h, 72h and 96h, observe the alteration under the contrast phase microscope. Positive control group CNTF(10-4g/L) and blank control group were set up and observation timing point were same as above. In each control groups and experimental groups 12 wells cells were observed and each well was repeatedly observed 3 times. The observation indexes were prominence numbers & axonal length of rat RGCs and the analyses software was IPP image analytical system.
2. Investigation of LEDGFp52 gene & protein regulating RGC neuronal specificity associated gene & protein: the treatment factors and subgroups were same as above. The detection indexes were gene expression changes & protein expression variations of GAP-43, NF-L and MAP-2, the gene changing was studied by RT-PCR and the protein varying was detected by CMIFC. The analyses softwares were Quantity One & LSM510-Meta-Expert, the former was used in semiquantitative analysis relative amount of each gene in mRNA level and the latter was applied in semiquantitative analysis mean fluorescence intensity of each protein.
Results
1. The serum-free Neurobasal TM Media is a better culturing system for RGCs, both gene and protein of LEDGFp52 was expressed in RGCs.
2. LEDGFp52 gene was inserted into prokaryotic expression vector by gene recombination technique, the construction was identified by enzyme digesting and sequencing, and the target protein dissoluble expression was induced by IPTG, the expression amount of rhLEDGFp52 was accounted to 34.63% of total bacteria protein. The Westernblotting result revealed that rhLEDGFp52 protein could be specifically integrated with LEDGF monoclone antibody. rhLEDGFp52 was purified by Ni-NTA His.Bind.Resin Method, and the final concentration was reached to 520μg.mL-1, its purity was 87.93%.
3. LEDGFp52 gene fragment was successfully inserted into the adenoviral shuttle plasmid pAdTrack-CMV and pAdTrack-CMV-LEDGFp52 was obtained. Then, it was cotransfected BJ5183 bacteria with TyV adenoviral backbone plasmid pAdeasy-1, the recombinant adenoviral vector pAd-LEDGFp52 was produced by homologous recombination in bacteria. And, pAd-LEDGFp52 was transformed into 293cells with lipofectamine TM 2000, the recombinant adenovirus Ad-LEDGFp52 was bred, and the titre was upto 5×1012 pfu.L-1.At last, 293cells were transfected by Ad-LEDGFp52 in vitro, the expression of LEDGFp52 was confirmed by CPE and Westblotting.
4. LEDGFp52 gene RNA interference eukaryotic expression vector was successfully constructed, the alteration of LEDGFp52 protein expression was detected by Westblotting in 48h once recombinant plasmid transfected HeLa cells, and the expression was significantly downregulated about 70%.
5. The RGCs growth was regulated by LEDGFp52 gene as well as protein, and manifestation was as follows: the axonal length was increased by up-regulation, and decreased by down-regulation; the peak potency appeared in positive regulation.
6. LEDGFp52 was an arborisation factor as well as an axonal elongation factor of RGC.
7. The RGCs neuronal specificity growth associated genes & proteins—GAP-43 , NF-L and MAP-2 were noticeably regulated by LEDGFp52 gene & protein, and manifestation was as follows: all genes & proteins expression were heightened by up-regulation and depressed by down-regulation.
Conclusions
1. The expression of LEDGFp52 on RGCs was first established.
2. rhLEDGFp52 protein was successfully obtained, LEDGFp52 recombinant adenovirus was prepared and small interference RNA recombinant was constructed which could be used in suppressing expression of LEDGFp52. All the protein, virus and recombinant had provided material prerequisites for further studying on biological effect of LEDGFp52 on ocular region.
3.The process numbers & axonal length of RGCs were significantly impacted by LEDGFp52 gene & protein .And LEDGFp52 is much more competent than CNTF.
4. LEDGFp52 was an arborisation factor of RGCs as well as axonal elongation factor .
5. LEDGFp52 gene & protein noticeably regulated the RGCs neuronal specificity growth associated gene &proteins—GAP-43, NF-L and MAP-2 regulating RGCs growth. And this is the main mechanism of LEDGFp52 regulating RGCs growth.
6. There were some other arborisation factors participating in controlling RGC arborisation by LEDGFp52 gene.
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