UVB导致HaCat细胞凋亡机制的初步研究
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摘要
臭氧是由太阳辐射使氧分子分解后,一个氧原子和另一个氧分子结合而成,是地球大气层中的一种蓝色、有刺激性的微量气体,也是平流层大气的最关键组成组分。尽管臭氧层在地球表面只占大气层的百万分之几,却吸收了来自太阳99%的高强度紫外线(ultraviolet, UV),保护了人类和生物免遭紫外线的伤害。但是由于一些自然因素,以及一些人类活动对环境的影响,使地球上方的臭氧层遭到破坏继而出现大面积的臭氧空洞,臭氧空洞形成无疑会导致大气层对来自太阳的紫外线的阻挡作用大大减弱,使辐射到地球上的紫外线增强,从而对生物产生一些危害效应。2006年9月,南极臭氧空洞的面积达到2950万平方公里,总面积超过了3个中国,而且,据科学家们预计,在今后的10-20年中的时间内,臭氧空洞仍有可能恶化。虽然现在全球都在采取一些必要的措施如在制冷剂中使用氟利昂的替代品等的来努力减少对臭氧层的继续破坏,甚至在将来有可能使地球上方的臭氧层空洞得以修复,但是据世界卫生组织估计,地球上的动物、植物以及浮游生物等在未来的40-100年里还将会受到越来越多的紫外线特别是紫外线B的辐射作用,这不仅对人类的健康本身造成危害,如皮肤癌症、机体免疫的损害、晶体甚至视网膜的损伤等,而且会对人类的生存环境也能造成不可估量的影响。
来自太阳的紫外线按其波长的不同可分为UVA(波长：315-400nm)、UVB(波长：280-315nm)、UVC(波长：100-280nm)三种紫外辐射。由于大气层的阻挡作用,所有的UVC和大约90%的UVB能被臭氧、水汽、氧和二氧化碳吸收,UVA则较少受到大气的影响,因此,到达地面的紫外线主要是UVA和少量UVB。同时,地球上方的臭氧层对紫外线的吸收作用急剧下降的区域,为310nm-315nm的范围内,此即UVB的波长范围,也就是说,地球臭氧层的破坏,会导致到达地球表面的UVB的量迅速增加,因此UVB对生物体的损伤作用,越来越得到世界各国的关注。
人类的皮肤覆盖于机体表面,是抵抗外界损伤的第一道防线,会比其他器官接受更多来自外界环境的辐射例如紫外线及可见光的辐射。照射到皮肤的紫外线95%的被角质细胞所吸收。环境中UVB的增加,无疑会加重对皮肤角质细胞的损伤,例如引起皮肤红斑和炎症反应。因此,本实验中选用了人永生化上皮细胞(HaCat细胞)作为研究对象,以波长为310nm的UVB作为干预因子,深入研究UVB作用于细胞后经由多种信号转导通路而调节细胞周期的机制。以往的研究表明,UVB至少可以通过下述四种途径破坏细胞的正常功能、影响细胞的生存状况如导致细胞的坏死或凋亡等：1：对细胞DNA的直接损伤；2：激活神经鞘磷脂酶使之降解神经鞘磷脂,从而导致“第二信使”神经酰胺及其衍生物水平增加：3：活化细胞膜表面的死亡受体如CD95等；4：经由对细胞膜和线粒体膜的作用而产生自由基和脂质过氧化产物。那么,UVB作用于HaCat细胞,终究是通过哪些机制引起HaCat细胞产生损伤效应,涉及到MAPK信号途径的哪些通路呢,又有哪些信号分子涉及到其作用机制中呢?这成了我们的实验关注的一个重点。
本研究分三个阶段,首先在实验一中用不同剂量的UVB照射HaCat细胞,并在UVB照射后在不同的时间点,观察了细胞的活性、细胞凋亡的变化情况,并观察了p38、p42/p44、p53、PARP、14-3-3σ等分子的变化情况,然后根据实验一发现的14-3-3σ表达在UVB照后出现改变的现象,以及文献调研总结出的14-3-3σ在细胞凋亡／周期阻滞中的作用,认为为了今后进一步深入研究14-3-3σ在UVB导致的细胞信号通路中的作用,建立14-3-3σ蛋白干扰和高表达的HaCat细胞株是非常有必要的,因此进行了实验二和实验三的研究,即分别构建了14-3-3σ稳定干扰和稳定高表达的HaCat细胞株。
目的：
1、探讨p38信号途径是否参与了UVB导致的细胞凋亡及其作用机制；
2、探讨ERK信号途径是否参与了UVB导致的细胞凋亡；
3、探讨UVB照射后,14-3-3σmRNA表达情况；
4、构建14-3-3σ干扰逆转录病毒载体,建立其稳定转染的HaCat细胞系；
5、构建14-3-3σ逆转录病毒载体,并建立高表达14-3-3σ的HaCat细胞系。
方法：
本研究方法主要包括三部分。
第一部分
1、以HaCat细胞为实验材料,“MTT”法观察HaCat细胞受到lmin、3min、5min、10min、15min的UVB照射后,分别在照后Oh、3h、6h、9h、12h、18h、24h和30h的细胞存活率；
2、HaCat细胞接受假照、以及UVB分别照射HaCat细胞lmin、3min、5min、8min、10min,在照后4h、8h、12h、24h,用Hoechst33258染料对HaCat细胞进行染色,并用荧光显微镜观察HaCat细胞凋亡的情况；
3、5min的UVB射线照射HaCat细胞,在照后12h收集细胞,并用weatern blotting免疫印迹法观察p38、p44/p42、p53、PARP等蛋白的表达改变情况；
4、分别用不同剂量(假照、UVB照射lmin、3min、5min、8min、10min)的UVB照射HaCat细胞,在照后1h收集细胞和用5min的UVB照射HaCat细胞,在照后Oh、1h、2h、4h、6h、12h收集细胞,提取细胞的总RNA,并通过反转录PCR反转录成cDNA,用1%的琼脂糖凝胶电泳,观测14-3-3σmRNA表达情况的改变。
第二部分
5、通过GeneBank检索14-3-3σ的全长序列,遵循siRNA设计的原则,在线寻找了三个合适的干扰靶位点,分别针对这三个靶位点设计并合成三对ShRNA的干扰片段,并定向插入到pSuper-retro-EGFP/neo质粒中。
6、pSuper-retro-EGFP/neo质粒转化到大肠杆菌DH5α细菌体内进行质粒扩增,菌落PCR鉴定,筛选出的阳性克隆进行测序鉴定。
7、分别将测序结果显示正确的质粒通过LipofectamineTM2000瞬转HaCat细胞48h,通过western blotting免疫印迹法筛选出干扰效果最好的质粒,用该质粒转化STBL3菌株进一步进行质粒扩增。将获得的pSUPER-retro-EGFP/neo-si-14-3-3o质粒和PIK包装质粒、293FT细胞共培养进行病毒包装和扩增,获取逆转录病毒载体。
8、将获得的逆转录病毒载体感染HaCat细胞,共感染三次,经6418持续压力筛选后,获得14-3-3σ稳定干扰的HaCat细胞株。
9、为验证干扰效果,通过western blotting免疫印迹和实时荧光定量PCR的鉴定14-3-3σ的mRNA和蛋白的表达情况。
第三部分
10、首先通过对GeneBank提供的14-3-3σ基因全长序列进行的分析,发现14-3-3σ基因可用BglⅡ和BamHⅠ进行双酶切,并置于CMV IE启动子之后。因此选用pLEGFP-N1质粒作为空载质粒,并设计扩增14-3-3σ的上游引物和下游引物,该引物中同时包含BglⅡ和BamHⅠ酶切位点以及它们的保护碱基。
11、以人的基因组作为模版,选用高保真的DNA连接酶,通过普通PCR扩增出14-3-3σ基因的全长序列,将扩增产物通过1%的琼脂糖凝胶电泳进行鉴定,并用琼脂糖凝胶DNA回收试剂盒回收扩增的目的DNA片段。
12、把14-3-3σ基因的胶回收产物和pLEGFP-N1载体分别用BglⅡ和BamHⅠ进行双酶切后,用TaKaRa的T4 DNA连接酶进行连接,转化感受态的大肠杆菌DH5α,并进行菌落PCR酶切鉴定,阳性克隆测序。
13、将测序结果显示正确的pLEGFP-Nl-14-3-3σ质粒在STBL3菌株中进一步扩增。
14、pLEGFP-N1-14-3-3σ质粒和PIK包装质粒、293FT细胞共培养进行病毒包装、扩增和纯化,获取逆转录病毒载体。
15、将获得的逆转录病毒载体感染HaCat细胞,共感染三次,经G418持续压力筛选后,获得14-3-3σ高表达稳定细胞株。
16、为验证表达效果,通过western blotting免疫印迹和实时荧光定量PCR的鉴定14-3-3σ的mRNA和蛋白表达情况。
结果：
实验一部分：
1、UVB照射HaCat细胞,在剂量相同情况下,随着照射后培养细胞的时间延长,HaCat细胞存活率呈现先下降后上升的趋势,即下降到最低值后恢复。UVB照射haCat细胞剂量不同,但照后相同的培养时间的情况下,随着照射剂量的增加,HaCat细胞存活率逐渐减少,而且15min的UVB照射组,在照后30h仍未出现恢复。
2、不同的UVB照射剂量和UVB照射后不同时间之间,HaCat细胞的凋亡率均有显著差异(P<0.05)。UVB照射5min组的凋亡率在照后各个时间点均比UVB照射HaCat细胞0min、1min、3min组凋亡率高(P<0.05),和UVB照射8mmin、10min比,在UVB照后12h,UVB照射5min组的凋亡率比8min、10min组高,差异显著(P<0.05)。UVB照射5min组,在照后培养12h时,凋亡率最高。
3、p44/42的含量在UVB照后不同时间点没有明显变化；而p38含量在UVB照后4h呈现递增性增加,至24h仍然高表达；p53在UVB照射后呈递增性增加,至24h仍高表达；PARP在UVB照后呈递增性增加,在UVB照后12h含量下降,而其降解产物在照后同样呈递增性增加,至12h达高峰,24h时含量下降。
4、不同剂量,即假照组、UVB照射Hacat细胞1min、3min、5min、8min、10minUVB组,14-3-3σmRNA的表达在3min、5min、8min、10min照射组均见到表达量的明显增加;5minUVB照射,假照组和照后1h、2h、4h、6h、12h组相比,照后1h即有mRNA的表达增加。
实验二部分：
5、分别针对14-3-3σ基因的不同的靶位点,设计并合成了三条14-3-3σ的干扰序列,并筛选出干扰效果最好的一条序列。即针对14-3-3σ基因第195至第214位点设计的干扰序列RNAi-5'-ACCTGCTCTCAGTAGCCTA-3',干扰效果最好,其正义链为5'GATCCCC ACCTGCTCTCAGTAGCCTA TTCAAGAGA TAGGCTACTGAGAGCAGGT TTTTT A3',反义链为Sh-14-3-3σ-R3 5'AGCTT AAAAA ACCTGCTCTCAGTAGCCTA TCTCTTGAA TAGGCTACTGAGAGCAGGT GGG 3'。
6、干扰序列和pSUPER-retro-EGFP/neo质粒连接重组后,经过菌落PCR鉴定,筛选出阳性克隆,进一步的测序结果表明,成功合成了pSUPER-retro-EGFP/ neo-si14-3-3σ质粒。并和包装质粒PIK、293FT细胞共培养产生pSUPER-retroEGFP/neo-si14-3-3σ逆转录病毒载体。
7、pSUPER-retro-EGFP/neo-si14-3-3σ逆转录病毒载体稳定转染的HaCat细胞在倒置显微镜下呈绿色荧光,表明细胞中有外源性基因的表达。
8、通过实时荧光定量PCR检测,发现14-3-3σ稳定干扰HaCat细胞株和HaCat细胞株相比,其14-3-3σmRNA表达明显下降,平均抑制率达到70%以上。
9、通过weatern blotting免疫印迹检测稳定转染的HaCat细胞14-3-3σ蛋白的表达结果显示,稳定转染株和HaCat细胞株相比,14-3-3σ的表达明显降低。
实验三部分：
10、以人的基因组为模板,成功扩增出14-3-3σ的全长序列,并进行酶切鉴定,大小和目的基因相符。
11、将14-3-3σ基因和pLEGFP-N1质粒连接重组后,经菌落PCR鉴定筛选出阳性克隆,再进行酶切鉴定和测序鉴定,其结果都表明成功合成了pLEGFP-N1-14-3-3σ质粒。
12、pLEGFP-N1-14-3-3σ质粒,PIK包装质粒、293FT细胞共培养产生pLEGFP-N1-14-3-3σ逆转录病毒载体。
13、pLEGFP-N1-14-3-3σ逆转录病毒载体稳定转染的HaCat细胞在倒置显微镜下呈绿色荧光,表明细胞中有外源性基因的表达。
14、通过实时荧光定量PCR检测,发现14-3-3σ高表达HaCat细胞株和HaCat细胞株相比,其14-3-3σmRNA表达明显上调,平均增强效率达2.71倍。
15、通过weatern blotting免疫印迹检测稳定转染的HaCat细胞14-3-3σ蛋白的表达结果显示,14-3-3σ稳定转染的HaCat细胞株和HaCat细胞株相比,14-3-3σ的表达明显增强。
结论：
1、UVB照射,能引起HaCat细胞的生长抑制和细胞凋亡,且呈剂量依赖性和时间依赖性；
2、UVB诱导细胞凋亡的可能途径之一是通过p38信号途径,而不是ERK信号途径；
3、UVB照射HaCat细胞,能引起14-3-3σmRNA表达的增加；
4、成功构建了pSUPER-retro-EGFP/neo-si14-3-3σ逆转录病毒载体,并构建了其稳定转染的HaCat细胞系；
5、成功构建了pLEGFP-N1-14-3-3σ逆转录病毒载体,并构建了其稳定转染的HaCat细胞系。
Atmospheric ozone molecule is combinated by an oxygen molecule and an oxygen atom which comes from oxygen molecule decomposed production by solar radiation. Ozone layer is blue and irritant trace gas in atmosphere and is the critical composition of stratospheric air. Although Ozone layer is not thichkness, it can provid protection for human and other creature by the absorption of 99% high intensity solar ultraviolet radiation. Because of some nature factor and influence of human activities on the environment, the ozone layer above the earth was destoryed and then large area ozone hole presented. It is undoubtedly that ozone hole will significantly reduce ozone's effect of blocking solar ultraviolet radiation,and result in more ultraviolet radiation reaching earth surface. In september 2006,the Antarctic ozone hole reached 29.5 million square kilometers, with a total area of more than three of China. Moreover, according to scientists assessment that in the next 10 to 20 years, deterioration of the ozone hole is still possible. Although in the global, human are taking the necessary measures to try to reduce the continued destruction of ozone layer,such as the use of freon succedaneum, even in the future we may repair the ozone hole, WHO estimates that earth animals, plants and plankton, etc. still will be receive more and more ultraviolet radiation in particular ultraviolet B(UVB) in the next 40-100 years. This is not only harmful to human health itself, but also for the human living environment.
According to their different wavelength,ultraviolet radiation from the sun can be divided into three partment:UVA(wavelength:315nm～400nm), UVB (wavelength: 280nm～315nm), UVC (wavelength:100nm～280nm).Because of the prevention of atmosphere, all UVC and approximately 90% UVB can be absorbed by ozone, water vapor, oxygen and carbon dioxide,UVA is more less affected by the atmosphere. Therefore, the UV radiation reaching earth surface combine UVA and a small amount of UVB. Meanwhile, the absorption fucution of ozone layer is mainly in the region of 310nm-315nm, namely UVB wavelength range. That is, the destruction of the ozone layer will lead to UVB of reaching earth surface rapidly increase, so the effect of UVB damage to the living being give rise to more and more human attention on it.
Skin that cover the body surface is the first line of defense against outside injury, it will receive more external environment radiation than the other organs,such as ultraviolet and visible light radiation.95% UV irradiation reaching skin was absorbed by keratinocytes. It is undoubtedly that the increase of environment UVB will induce more damage to keratinocytes, such as causing skin erythema and inflammation. Therefore, this study selected the immortalized human epithelial cells (HaCat cells) and 310nm UVB to investigate the damage mechanism. Previous studies showed that, UVB at least has four ways to destroy cell normal functions, to affect cell survival conditions, such as lead to cell necrosis or apoptosis.1:The direct damage to cell DNA;2:Activation of sphingomyelinase which can lead to sphingomyelin degradation, result in the increase level of ceramide and their derivatives;3:Activation of cell surface death receptors such as CD95, etc.; 4:Create free radicals and lipid peroxidation through the cell membrane and mitochondria membrane. Which pathways and signaling molecules involve in the damages of HaCat cell caused by UVB are still unknown. These are our focus in this study.
This study was divided into three stages. First, we observed the changes of cytoactive, apoptosis,p38, p42/p44, p53, PARP and 14-3-3σafter HaCat cell exposed different UVB doses and/or in different times after particular UVB dose. According to these discovers of 14-3-3σexpression changes after exposed to UVB in the first experiment and the important role of 14-3-3σin apoptosis and cell cycle arrest, we concluded that it is necessary for us to construct stably 14-3-3σRNAi HaCat cell lines and 14-3-3σstably over expression HaCat cell line for the further studies。
OBJECTIVE:
1、To explore the roles of p38 signal transduction pathway in UVB induced apoptosis;
2、To explore the roles of ERK signal transduction pathway in UVB induced apoptosis.
3、To investigate the changes of 14-3-3amRNA after expose to UVB irradiation;
4、To construct RNAi retroviral vector targeting 14-3-3σand establish stably transfected HaCat cell lines.
5、To construct thel4-3-3σretroviral vector and establish stably 14-3-3σover expression HaCat cell lines.
METHODS:
The research methods consists of three parts.
Part one
1、HaCat cells were used in this study. With 1min,3min,5min,10min and 15min UVB irradiation respectively, the ratio of cell survival after expose to UVB 0h,3h,6h,9h,12h,18h,24h and 30h was measured by MTT reduction assay.
2、After being irradiated by UVB radiation at the dosage of mock irradiation,1min,3min,5min,8min,10min respectively, apoptotic cells stained by Hoechst33258 were observed and counted through fluorescent microscope at the time points after irradiated 4h、8h、12h、24h.
3、After being irradiated by UVB radiation at the dosage of 5min UVB, western blotting was used to examine the expressions of p38,p42/p44,p53,PARP, and so on the time point after irradiated 12h.
4、After being irradiated by UVB radiation at the dosage of mock irradiation, lmin,3min,5min,8min and 10min UVB irradiation respectively, cells were collected on the time point after irradiated 1h.With 5min of UVB irradiation, the cells were collected on the time points after irradiated 0h,1h,2h,4h,6h,12h.The RNA were extracted from above groups and reversed into cDNA by reverse transcription-PCR. The 14-3-3a mRNA expressions were detected by 1% agarose gel electrophoresis. Part two
5、According to the 14-3-3σfull sequence searched by GeneBank and the principle of siRNA design,we searched three suitable RNAi target sites and chemically synthesized three pairs of hairpin siRNA of 14-3-3σ.And then three pairs of hairpin siRNA were inserted into pSUPER-retro-EGFP/neo plasmid.
6、pSUPER-retro-EGFP/neo-si14-3-3σwere transformed into competent DH5a cells to amplification itself. And then the positive clones were confirmed by sequencing.
7、The plasmids were transient transfected into HaCat cell by Lipofectamine TM2000 for 48h. The third hairpin siRNAi is the best one which were detected by western blotting.Therefore, the plasmids that contain the third hairpin siRNA were transformed into competent STBL3 cells for more amplification and then transfected into the packaging 293FT cells to generate, amplificate and depurate virus.
8、HaCat cells were infected with the recombinant retroviral vector three times。After sustained pressure by G418 selection, we construct stably 14-3-3a RNAi HaCat cell lines.
9、The expression of 14-3-3σwas detected by Western blotting and Real-time fluorescence quantitative PCR.
Part three
10、According to the analysis of 14-3-3σgene sequence,we found that 14-3-3σgene can be digested by Bgl II and BamH I,and can be placed into the sites after the CMV IE promoter. To amplification 14-3-3σgene, pLEGFP-N1 plasmid was selected as no-load plasmid in this study. The upstream primer and downstream primer contain there protective basic of 14-3-3σgene were then designed.
11、Useing the human genome as a template and high-fidelity DNA ligase, we amplified 14-3-3σgene by PCR. The PCR products were identified by 1% agarose gel electrophoresis and reclaimed amplified DNA fragments by agarose gel DNA extraction kit.
12、The productions of 14-3-3σsequence and pLEGFP-N1 plasmids were digested by Bgl II and BamH I. And then we used T4 DNA ligase to connect them. The connections were transformed into competent DH5a cells to amplificat connections. Then the positive clones were confirmed by sequencing.
13、pLEGFP-N1-14-3-3σplasmids were transformed into competent STBL3 cells for more amplification.
14、pLEGFP-N1-14-3-3σplasmids were transfected into the packaging 293FT cells to generated, amplificated and depurated virus.
15、HaCat cells were infected with the recombinant retroviral vectors three times.After selected by sustained pressure of G418, we constructed stably 14-3-3σover expression HaCat cell lines.
16、The expression of 14-3-3σwas detected by Western blotting and Real-time fluorescence quantitative PCR.
RESULTS:
Part one
1、At a fixed UVB irradiation dose, prolonged incubation of the cells following the irradiation resulted in decreased cell survival ratio, which, however, began to increased when the minimum rate was reached; Increased with UVB irradiated doses, the survival rate were taper off when HaCat cells were detched in same time. Further more, in the group with 15min UVB irradiated, there was no recover until 30h after irradiated.
2、There were significant deviation in apoptosis ratio among different dosages UVB irradiated and different time points after UVB irradiated(P<0.05). In differents dosages, the apoptosis ratio is the highest when HaCat cells were irradiated by UVB in five minutes, moreover the ratio peak after HaCat cells incubated for 12h.
3、There is no marked changes of p42/44 detected among the time points after UVB irradiation, meanwhile, the p38,p53 levels showed progressive increase after 4h, the expression of PARP also showed progressive increase, but the expression descended at the point of 12h after UVB irradiation, meanwhile cleaved PARP showed progressive increase and up to the peak at the point of 12h,and declined in 24h.
4、The 14-3-3σmRNA expression level was up-regulated at 3min,5min,8min and 10min UVB irradiated groups.With fixed dosage of 5min UVB irradiation, 14-3-3σmRNA expression level was up-regulated in lh after UVB irradiation. Part two
5、Design and synthesis three RNAi sequences to aim directly at different target sites of 14-3-3σsequence.And then selected the best one.it is RNAi-5'-ACCTGCTCTCAGTAGCCTA-3'.
Sh-14-3-3σ-F3 5'GATCCCC ACCTGCTCTCAGTAGCCTA TTCAAGAGA TAGGCTACTGAGAGCAGGT TTTTT A3'
Sh-14-3-3σ-R3 5'AGCTT AAAAA ACCTGCTCTCAGTAGCCTA TCTCTTGAA TAGGCTACTGAGAGCAGGT GGG 3'
6、The RNAi sequences and pSUPER-retro-EGFP/neo plasmids were recombined,the positive clone were identified by colony PCR screening, sequencing result showed that the recombinant PSuper-retro-EGFP/neo-si14-3-3σplasmid was successfully constructed. PSuper-retro-EGFP/neo-si14-3-3σplasmids,packaging plasmid PIK and 293 FT cells co-cultured to produce recombinant retroviral vector of PSuper-retro-neo-EGFP-si14-3-3σ.
7、Green fluorescence of the stable transfected HaCat cell lines could be observed under inverted fluorescence microscope.
8、The results of Real-time fluorescence quantitative PCR showed that the sequence of RNAi-14-3-3a could effectively down-regulate the level of 14-3-3σ, the average inhibition ratio over 70%.
9、The results of western blotting showed that the expression of 14-3-3σprotein was effectively down-regulated. Part Three
10、14-3-3σfull sequence was successfully amplified using template of human genome. The molecular weight of amplified DNA fragment was proved correct by 1% agarose gel electrophoresis.
11、The 14-3-3σsequence and pLEGFP-N1 plasmid were recombined, positive clone were identified by colony PCR screening,1% agarose gel electrophoresis, sequencing.All results showed that the recombinant pLEGFP-N1-14-3-3σplasmid was successfully constructed.
12、pLEGFP-N1-14-3-3σplasmids,packaging plasmids PIK and 293FT cells co-cultured to produce recombinant retroviral vectors of pLEGFP-N1-14-3-3σ.
13、Green fluorescence of the stable transfected HaCat cell lines could be observed under inverted fluorescence microscope. The result showed that exogenous gene can express in HaCat cell。
14The results of Real-time fluorescence quantitative PCR showed that the exogenous sequence could effectively up-regulate the level of 14-3-3σ.The stably transfection cell has 2.71-fold expression in average than normal cells。
15、The results of western blotting showed that the expression of 14-3-3σprotein was effectively up-regulated.
CONCLUSIONS:
1、UVB irradiation can induce HaCat cells growth inhibiting and apoptosis in a dose-and time-dependent manner;
2σThe UVB induced apoptosis may through by p38 pathway, not by ERK pathway.
3、14-3-3σmRNA expression was up-regulated by UVB irradiation.
4、We have successfully constructed the RNAi retroviral vector targeting 14-3-3a and have established stably transfected HaCat cell lines.
5、We have successfully constructed thel4-3-3σretroviral vector and have established stably transfected HaCat cell lines.
来自太阳的紫外线按其波长的不同可分为UVA(波长：315-400nm)、UVB(波长：280-315nm)、UVC(波长：100-280nm)三种紫外辐射。由于大气层的阻挡作用,所有的UVC和大约90%的UVB能被臭氧、水汽、氧和二氧化碳吸收,UVA则较少受到大气的影响,因此,到达地面的紫外线主要是UVA和少量UVB。同时,地球上方的臭氧层对紫外线的吸收作用急剧下降的区域,为310nm-315nm的范围内,此即UVB的波长范围,也就是说,地球臭氧层的破坏,会导致到达地球表面的UVB的量迅速增加,因此UVB对生物体的损伤作用,越来越得到世界各国的关注。
人类的皮肤覆盖于机体表面,是抵抗外界损伤的第一道防线,会比其他器官接受更多来自外界环境的辐射例如紫外线及可见光的辐射。照射到皮肤的紫外线95%的被角质细胞所吸收。环境中UVB的增加,无疑会加重对皮肤角质细胞的损伤,例如引起皮肤红斑和炎症反应。因此,本实验中选用了人永生化上皮细胞(HaCat细胞)作为研究对象,以波长为310nm的UVB作为干预因子,深入研究UVB作用于细胞后经由多种信号转导通路而调节细胞周期的机制。以往的研究表明,UVB至少可以通过下述四种途径破坏细胞的正常功能、影响细胞的生存状况如导致细胞的坏死或凋亡等：1：对细胞DNA的直接损伤；2：激活神经鞘磷脂酶使之降解神经鞘磷脂,从而导致“第二信使”神经酰胺及其衍生物水平增加：3：活化细胞膜表面的死亡受体如CD95等；4：经由对细胞膜和线粒体膜的作用而产生自由基和脂质过氧化产物。那么,UVB作用于HaCat细胞,终究是通过哪些机制引起HaCat细胞产生损伤效应,涉及到MAPK信号途径的哪些通路呢,又有哪些信号分子涉及到其作用机制中呢?这成了我们的实验关注的一个重点。
本研究分三个阶段,首先在实验一中用不同剂量的UVB照射HaCat细胞,并在UVB照射后在不同的时间点,观察了细胞的活性、细胞凋亡的变化情况,并观察了p38、p42/p44、p53、PARP、14-3-3σ等分子的变化情况,然后根据实验一发现的14-3-3σ表达在UVB照后出现改变的现象,以及文献调研总结出的14-3-3σ在细胞凋亡／周期阻滞中的作用,认为为了今后进一步深入研究14-3-3σ在UVB导致的细胞信号通路中的作用,建立14-3-3σ蛋白干扰和高表达的HaCat细胞株是非常有必要的,因此进行了实验二和实验三的研究,即分别构建了14-3-3σ稳定干扰和稳定高表达的HaCat细胞株。
目的：
1、探讨p38信号途径是否参与了UVB导致的细胞凋亡及其作用机制；
2、探讨ERK信号途径是否参与了UVB导致的细胞凋亡；
3、探讨UVB照射后,14-3-3σmRNA表达情况；
4、构建14-3-3σ干扰逆转录病毒载体,建立其稳定转染的HaCat细胞系；
5、构建14-3-3σ逆转录病毒载体,并建立高表达14-3-3σ的HaCat细胞系。
方法：
本研究方法主要包括三部分。
第一部分
1、以HaCat细胞为实验材料,“MTT”法观察HaCat细胞受到lmin、3min、5min、10min、15min的UVB照射后,分别在照后Oh、3h、6h、9h、12h、18h、24h和30h的细胞存活率；
2、HaCat细胞接受假照、以及UVB分别照射HaCat细胞lmin、3min、5min、8min、10min,在照后4h、8h、12h、24h,用Hoechst33258染料对HaCat细胞进行染色,并用荧光显微镜观察HaCat细胞凋亡的情况；
3、5min的UVB射线照射HaCat细胞,在照后12h收集细胞,并用weatern blotting免疫印迹法观察p38、p44/p42、p53、PARP等蛋白的表达改变情况；
4、分别用不同剂量(假照、UVB照射lmin、3min、5min、8min、10min)的UVB照射HaCat细胞,在照后1h收集细胞和用5min的UVB照射HaCat细胞,在照后Oh、1h、2h、4h、6h、12h收集细胞,提取细胞的总RNA,并通过反转录PCR反转录成cDNA,用1%的琼脂糖凝胶电泳,观测14-3-3σmRNA表达情况的改变。
第二部分
5、通过GeneBank检索14-3-3σ的全长序列,遵循siRNA设计的原则,在线寻找了三个合适的干扰靶位点,分别针对这三个靶位点设计并合成三对ShRNA的干扰片段,并定向插入到pSuper-retro-EGFP/neo质粒中。
6、pSuper-retro-EGFP/neo质粒转化到大肠杆菌DH5α细菌体内进行质粒扩增,菌落PCR鉴定,筛选出的阳性克隆进行测序鉴定。
7、分别将测序结果显示正确的质粒通过LipofectamineTM2000瞬转HaCat细胞48h,通过western blotting免疫印迹法筛选出干扰效果最好的质粒,用该质粒转化STBL3菌株进一步进行质粒扩增。将获得的pSUPER-retro-EGFP/neo-si-14-3-3o质粒和PIK包装质粒、293FT细胞共培养进行病毒包装和扩增,获取逆转录病毒载体。
8、将获得的逆转录病毒载体感染HaCat细胞,共感染三次,经6418持续压力筛选后,获得14-3-3σ稳定干扰的HaCat细胞株。
9、为验证干扰效果,通过western blotting免疫印迹和实时荧光定量PCR的鉴定14-3-3σ的mRNA和蛋白的表达情况。
第三部分
10、首先通过对GeneBank提供的14-3-3σ基因全长序列进行的分析,发现14-3-3σ基因可用BglⅡ和BamHⅠ进行双酶切,并置于CMV IE启动子之后。因此选用pLEGFP-N1质粒作为空载质粒,并设计扩增14-3-3σ的上游引物和下游引物,该引物中同时包含BglⅡ和BamHⅠ酶切位点以及它们的保护碱基。
11、以人的基因组作为模版,选用高保真的DNA连接酶,通过普通PCR扩增出14-3-3σ基因的全长序列,将扩增产物通过1%的琼脂糖凝胶电泳进行鉴定,并用琼脂糖凝胶DNA回收试剂盒回收扩增的目的DNA片段。
12、把14-3-3σ基因的胶回收产物和pLEGFP-N1载体分别用BglⅡ和BamHⅠ进行双酶切后,用TaKaRa的T4 DNA连接酶进行连接,转化感受态的大肠杆菌DH5α,并进行菌落PCR酶切鉴定,阳性克隆测序。
13、将测序结果显示正确的pLEGFP-Nl-14-3-3σ质粒在STBL3菌株中进一步扩增。
14、pLEGFP-N1-14-3-3σ质粒和PIK包装质粒、293FT细胞共培养进行病毒包装、扩增和纯化,获取逆转录病毒载体。
15、将获得的逆转录病毒载体感染HaCat细胞,共感染三次,经G418持续压力筛选后,获得14-3-3σ高表达稳定细胞株。
16、为验证表达效果,通过western blotting免疫印迹和实时荧光定量PCR的鉴定14-3-3σ的mRNA和蛋白表达情况。
结果：
实验一部分：
1、UVB照射HaCat细胞,在剂量相同情况下,随着照射后培养细胞的时间延长,HaCat细胞存活率呈现先下降后上升的趋势,即下降到最低值后恢复。UVB照射haCat细胞剂量不同,但照后相同的培养时间的情况下,随着照射剂量的增加,HaCat细胞存活率逐渐减少,而且15min的UVB照射组,在照后30h仍未出现恢复。
2、不同的UVB照射剂量和UVB照射后不同时间之间,HaCat细胞的凋亡率均有显著差异(P<0.05)。UVB照射5min组的凋亡率在照后各个时间点均比UVB照射HaCat细胞0min、1min、3min组凋亡率高(P<0.05),和UVB照射8mmin、10min比,在UVB照后12h,UVB照射5min组的凋亡率比8min、10min组高,差异显著(P<0.05)。UVB照射5min组,在照后培养12h时,凋亡率最高。
3、p44/42的含量在UVB照后不同时间点没有明显变化；而p38含量在UVB照后4h呈现递增性增加,至24h仍然高表达；p53在UVB照射后呈递增性增加,至24h仍高表达；PARP在UVB照后呈递增性增加,在UVB照后12h含量下降,而其降解产物在照后同样呈递增性增加,至12h达高峰,24h时含量下降。
4、不同剂量,即假照组、UVB照射Hacat细胞1min、3min、5min、8min、10minUVB组,14-3-3σmRNA的表达在3min、5min、8min、10min照射组均见到表达量的明显增加;5minUVB照射,假照组和照后1h、2h、4h、6h、12h组相比,照后1h即有mRNA的表达增加。
实验二部分：
5、分别针对14-3-3σ基因的不同的靶位点,设计并合成了三条14-3-3σ的干扰序列,并筛选出干扰效果最好的一条序列。即针对14-3-3σ基因第195至第214位点设计的干扰序列RNAi-5'-ACCTGCTCTCAGTAGCCTA-3',干扰效果最好,其正义链为5'GATCCCC ACCTGCTCTCAGTAGCCTA TTCAAGAGA TAGGCTACTGAGAGCAGGT TTTTT A3',反义链为Sh-14-3-3σ-R3 5'AGCTT AAAAA ACCTGCTCTCAGTAGCCTA TCTCTTGAA TAGGCTACTGAGAGCAGGT GGG 3'。
6、干扰序列和pSUPER-retro-EGFP/neo质粒连接重组后,经过菌落PCR鉴定,筛选出阳性克隆,进一步的测序结果表明,成功合成了pSUPER-retro-EGFP/ neo-si14-3-3σ质粒。并和包装质粒PIK、293FT细胞共培养产生pSUPER-retroEGFP/neo-si14-3-3σ逆转录病毒载体。
7、pSUPER-retro-EGFP/neo-si14-3-3σ逆转录病毒载体稳定转染的HaCat细胞在倒置显微镜下呈绿色荧光,表明细胞中有外源性基因的表达。
8、通过实时荧光定量PCR检测,发现14-3-3σ稳定干扰HaCat细胞株和HaCat细胞株相比,其14-3-3σmRNA表达明显下降,平均抑制率达到70%以上。
9、通过weatern blotting免疫印迹检测稳定转染的HaCat细胞14-3-3σ蛋白的表达结果显示,稳定转染株和HaCat细胞株相比,14-3-3σ的表达明显降低。
实验三部分：
10、以人的基因组为模板,成功扩增出14-3-3σ的全长序列,并进行酶切鉴定,大小和目的基因相符。
11、将14-3-3σ基因和pLEGFP-N1质粒连接重组后,经菌落PCR鉴定筛选出阳性克隆,再进行酶切鉴定和测序鉴定,其结果都表明成功合成了pLEGFP-N1-14-3-3σ质粒。
12、pLEGFP-N1-14-3-3σ质粒,PIK包装质粒、293FT细胞共培养产生pLEGFP-N1-14-3-3σ逆转录病毒载体。
13、pLEGFP-N1-14-3-3σ逆转录病毒载体稳定转染的HaCat细胞在倒置显微镜下呈绿色荧光,表明细胞中有外源性基因的表达。
14、通过实时荧光定量PCR检测,发现14-3-3σ高表达HaCat细胞株和HaCat细胞株相比,其14-3-3σmRNA表达明显上调,平均增强效率达2.71倍。
15、通过weatern blotting免疫印迹检测稳定转染的HaCat细胞14-3-3σ蛋白的表达结果显示,14-3-3σ稳定转染的HaCat细胞株和HaCat细胞株相比,14-3-3σ的表达明显增强。
结论：
1、UVB照射,能引起HaCat细胞的生长抑制和细胞凋亡,且呈剂量依赖性和时间依赖性；
2、UVB诱导细胞凋亡的可能途径之一是通过p38信号途径,而不是ERK信号途径；
3、UVB照射HaCat细胞,能引起14-3-3σmRNA表达的增加；
4、成功构建了pSUPER-retro-EGFP/neo-si14-3-3σ逆转录病毒载体,并构建了其稳定转染的HaCat细胞系；
5、成功构建了pLEGFP-N1-14-3-3σ逆转录病毒载体,并构建了其稳定转染的HaCat细胞系。
Atmospheric ozone molecule is combinated by an oxygen molecule and an oxygen atom which comes from oxygen molecule decomposed production by solar radiation. Ozone layer is blue and irritant trace gas in atmosphere and is the critical composition of stratospheric air. Although Ozone layer is not thichkness, it can provid protection for human and other creature by the absorption of 99% high intensity solar ultraviolet radiation. Because of some nature factor and influence of human activities on the environment, the ozone layer above the earth was destoryed and then large area ozone hole presented. It is undoubtedly that ozone hole will significantly reduce ozone's effect of blocking solar ultraviolet radiation,and result in more ultraviolet radiation reaching earth surface. In september 2006,the Antarctic ozone hole reached 29.5 million square kilometers, with a total area of more than three of China. Moreover, according to scientists assessment that in the next 10 to 20 years, deterioration of the ozone hole is still possible. Although in the global, human are taking the necessary measures to try to reduce the continued destruction of ozone layer,such as the use of freon succedaneum, even in the future we may repair the ozone hole, WHO estimates that earth animals, plants and plankton, etc. still will be receive more and more ultraviolet radiation in particular ultraviolet B(UVB) in the next 40-100 years. This is not only harmful to human health itself, but also for the human living environment.
According to their different wavelength,ultraviolet radiation from the sun can be divided into three partment:UVA(wavelength:315nm～400nm), UVB (wavelength: 280nm～315nm), UVC (wavelength:100nm～280nm).Because of the prevention of atmosphere, all UVC and approximately 90% UVB can be absorbed by ozone, water vapor, oxygen and carbon dioxide,UVA is more less affected by the atmosphere. Therefore, the UV radiation reaching earth surface combine UVA and a small amount of UVB. Meanwhile, the absorption fucution of ozone layer is mainly in the region of 310nm-315nm, namely UVB wavelength range. That is, the destruction of the ozone layer will lead to UVB of reaching earth surface rapidly increase, so the effect of UVB damage to the living being give rise to more and more human attention on it.
Skin that cover the body surface is the first line of defense against outside injury, it will receive more external environment radiation than the other organs,such as ultraviolet and visible light radiation.95% UV irradiation reaching skin was absorbed by keratinocytes. It is undoubtedly that the increase of environment UVB will induce more damage to keratinocytes, such as causing skin erythema and inflammation. Therefore, this study selected the immortalized human epithelial cells (HaCat cells) and 310nm UVB to investigate the damage mechanism. Previous studies showed that, UVB at least has four ways to destroy cell normal functions, to affect cell survival conditions, such as lead to cell necrosis or apoptosis.1:The direct damage to cell DNA;2:Activation of sphingomyelinase which can lead to sphingomyelin degradation, result in the increase level of ceramide and their derivatives;3:Activation of cell surface death receptors such as CD95, etc.; 4:Create free radicals and lipid peroxidation through the cell membrane and mitochondria membrane. Which pathways and signaling molecules involve in the damages of HaCat cell caused by UVB are still unknown. These are our focus in this study.
This study was divided into three stages. First, we observed the changes of cytoactive, apoptosis,p38, p42/p44, p53, PARP and 14-3-3σafter HaCat cell exposed different UVB doses and/or in different times after particular UVB dose. According to these discovers of 14-3-3σexpression changes after exposed to UVB in the first experiment and the important role of 14-3-3σin apoptosis and cell cycle arrest, we concluded that it is necessary for us to construct stably 14-3-3σRNAi HaCat cell lines and 14-3-3σstably over expression HaCat cell line for the further studies。
OBJECTIVE:
1、To explore the roles of p38 signal transduction pathway in UVB induced apoptosis;
2、To explore the roles of ERK signal transduction pathway in UVB induced apoptosis.
3、To investigate the changes of 14-3-3amRNA after expose to UVB irradiation;
4、To construct RNAi retroviral vector targeting 14-3-3σand establish stably transfected HaCat cell lines.
5、To construct thel4-3-3σretroviral vector and establish stably 14-3-3σover expression HaCat cell lines.
METHODS:
The research methods consists of three parts.
Part one
1、HaCat cells were used in this study. With 1min,3min,5min,10min and 15min UVB irradiation respectively, the ratio of cell survival after expose to UVB 0h,3h,6h,9h,12h,18h,24h and 30h was measured by MTT reduction assay.
2、After being irradiated by UVB radiation at the dosage of mock irradiation,1min,3min,5min,8min,10min respectively, apoptotic cells stained by Hoechst33258 were observed and counted through fluorescent microscope at the time points after irradiated 4h、8h、12h、24h.
3、After being irradiated by UVB radiation at the dosage of 5min UVB, western blotting was used to examine the expressions of p38,p42/p44,p53,PARP, and so on the time point after irradiated 12h.
4、After being irradiated by UVB radiation at the dosage of mock irradiation, lmin,3min,5min,8min and 10min UVB irradiation respectively, cells were collected on the time point after irradiated 1h.With 5min of UVB irradiation, the cells were collected on the time points after irradiated 0h,1h,2h,4h,6h,12h.The RNA were extracted from above groups and reversed into cDNA by reverse transcription-PCR. The 14-3-3a mRNA expressions were detected by 1% agarose gel electrophoresis. Part two
5、According to the 14-3-3σfull sequence searched by GeneBank and the principle of siRNA design,we searched three suitable RNAi target sites and chemically synthesized three pairs of hairpin siRNA of 14-3-3σ.And then three pairs of hairpin siRNA were inserted into pSUPER-retro-EGFP/neo plasmid.
6、pSUPER-retro-EGFP/neo-si14-3-3σwere transformed into competent DH5a cells to amplification itself. And then the positive clones were confirmed by sequencing.
7、The plasmids were transient transfected into HaCat cell by Lipofectamine TM2000 for 48h. The third hairpin siRNAi is the best one which were detected by western blotting.Therefore, the plasmids that contain the third hairpin siRNA were transformed into competent STBL3 cells for more amplification and then transfected into the packaging 293FT cells to generate, amplificate and depurate virus.
8、HaCat cells were infected with the recombinant retroviral vector three times。After sustained pressure by G418 selection, we construct stably 14-3-3a RNAi HaCat cell lines.
9、The expression of 14-3-3σwas detected by Western blotting and Real-time fluorescence quantitative PCR.
Part three
10、According to the analysis of 14-3-3σgene sequence,we found that 14-3-3σgene can be digested by Bgl II and BamH I,and can be placed into the sites after the CMV IE promoter. To amplification 14-3-3σgene, pLEGFP-N1 plasmid was selected as no-load plasmid in this study. The upstream primer and downstream primer contain there protective basic of 14-3-3σgene were then designed.
11、Useing the human genome as a template and high-fidelity DNA ligase, we amplified 14-3-3σgene by PCR. The PCR products were identified by 1% agarose gel electrophoresis and reclaimed amplified DNA fragments by agarose gel DNA extraction kit.
12、The productions of 14-3-3σsequence and pLEGFP-N1 plasmids were digested by Bgl II and BamH I. And then we used T4 DNA ligase to connect them. The connections were transformed into competent DH5a cells to amplificat connections. Then the positive clones were confirmed by sequencing.
13、pLEGFP-N1-14-3-3σplasmids were transformed into competent STBL3 cells for more amplification.
14、pLEGFP-N1-14-3-3σplasmids were transfected into the packaging 293FT cells to generated, amplificated and depurated virus.
15、HaCat cells were infected with the recombinant retroviral vectors three times.After selected by sustained pressure of G418, we constructed stably 14-3-3σover expression HaCat cell lines.
16、The expression of 14-3-3σwas detected by Western blotting and Real-time fluorescence quantitative PCR.
RESULTS:
Part one
1、At a fixed UVB irradiation dose, prolonged incubation of the cells following the irradiation resulted in decreased cell survival ratio, which, however, began to increased when the minimum rate was reached; Increased with UVB irradiated doses, the survival rate were taper off when HaCat cells were detched in same time. Further more, in the group with 15min UVB irradiated, there was no recover until 30h after irradiated.
2、There were significant deviation in apoptosis ratio among different dosages UVB irradiated and different time points after UVB irradiated(P<0.05). In differents dosages, the apoptosis ratio is the highest when HaCat cells were irradiated by UVB in five minutes, moreover the ratio peak after HaCat cells incubated for 12h.
3、There is no marked changes of p42/44 detected among the time points after UVB irradiation, meanwhile, the p38,p53 levels showed progressive increase after 4h, the expression of PARP also showed progressive increase, but the expression descended at the point of 12h after UVB irradiation, meanwhile cleaved PARP showed progressive increase and up to the peak at the point of 12h,and declined in 24h.
4、The 14-3-3σmRNA expression level was up-regulated at 3min,5min,8min and 10min UVB irradiated groups.With fixed dosage of 5min UVB irradiation, 14-3-3σmRNA expression level was up-regulated in lh after UVB irradiation. Part two
5、Design and synthesis three RNAi sequences to aim directly at different target sites of 14-3-3σsequence.And then selected the best one.it is RNAi-5'-ACCTGCTCTCAGTAGCCTA-3'.
Sh-14-3-3σ-F3 5'GATCCCC ACCTGCTCTCAGTAGCCTA TTCAAGAGA TAGGCTACTGAGAGCAGGT TTTTT A3'
Sh-14-3-3σ-R3 5'AGCTT AAAAA ACCTGCTCTCAGTAGCCTA TCTCTTGAA TAGGCTACTGAGAGCAGGT GGG 3'
6、The RNAi sequences and pSUPER-retro-EGFP/neo plasmids were recombined,the positive clone were identified by colony PCR screening, sequencing result showed that the recombinant PSuper-retro-EGFP/neo-si14-3-3σplasmid was successfully constructed. PSuper-retro-EGFP/neo-si14-3-3σplasmids,packaging plasmid PIK and 293 FT cells co-cultured to produce recombinant retroviral vector of PSuper-retro-neo-EGFP-si14-3-3σ.
7、Green fluorescence of the stable transfected HaCat cell lines could be observed under inverted fluorescence microscope.
8、The results of Real-time fluorescence quantitative PCR showed that the sequence of RNAi-14-3-3a could effectively down-regulate the level of 14-3-3σ, the average inhibition ratio over 70%.
9、The results of western blotting showed that the expression of 14-3-3σprotein was effectively down-regulated. Part Three
10、14-3-3σfull sequence was successfully amplified using template of human genome. The molecular weight of amplified DNA fragment was proved correct by 1% agarose gel electrophoresis.
11、The 14-3-3σsequence and pLEGFP-N1 plasmid were recombined, positive clone were identified by colony PCR screening,1% agarose gel electrophoresis, sequencing.All results showed that the recombinant pLEGFP-N1-14-3-3σplasmid was successfully constructed.
12、pLEGFP-N1-14-3-3σplasmids,packaging plasmids PIK and 293FT cells co-cultured to produce recombinant retroviral vectors of pLEGFP-N1-14-3-3σ.
13、Green fluorescence of the stable transfected HaCat cell lines could be observed under inverted fluorescence microscope. The result showed that exogenous gene can express in HaCat cell。
14The results of Real-time fluorescence quantitative PCR showed that the exogenous sequence could effectively up-regulate the level of 14-3-3σ.The stably transfection cell has 2.71-fold expression in average than normal cells。
15、The results of western blotting showed that the expression of 14-3-3σprotein was effectively up-regulated.
CONCLUSIONS:
1、UVB irradiation can induce HaCat cells growth inhibiting and apoptosis in a dose-and time-dependent manner;
2σThe UVB induced apoptosis may through by p38 pathway, not by ERK pathway.
3、14-3-3σmRNA expression was up-regulated by UVB irradiation.
4、We have successfully constructed the RNAi retroviral vector targeting 14-3-3a and have established stably transfected HaCat cell lines.
5、We have successfully constructed thel4-3-3σretroviral vector and have established stably transfected HaCat cell lines.
引文
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