铅诱导PC12细胞旁观者效应及细胞间隙连接通讯（GJIC）作用研究
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摘要
研究背景
铅是工业上常用的重金属之一,可以通过呼吸和消化系统进入人体。铅具有很强的神经毒性,可导致不可逆的神经系统损伤,儿童在发育期对铅暴露产生的毒性尤为敏感。科学家们发现即使儿童的血铅浓度保持在可接受水平,儿童的智力发育仍会受到明显影响。因此,铅的神经毒性受到了广泛关注。
细胞生长的微环境是保持细胞正常增殖、分化、代谢和功能活动的重要条件。细胞对外界的微环境非常敏感,在同一共培养体系中,细胞之间或细胞与胞外基质之间的直接接触可以影响细胞的生长状态。近几十年来,在放射生物学领域发现,当少数细胞受到射线照射时,没有受到射线直接照射的周围细胞也能表现出一些损伤效应。大量研究已经证明在体内和体外实验中确实存在损伤传递效应。例如,慢性粒细胞白血病患儿的脾部在接受放射线照射治疗时,胸骨骨髓细胞发生了损伤；局部氧化损伤可以通过GJIC在内皮细胞之间传递。有人提出用肿瘤自杀基因治疗肿瘤,当部分肿瘤细胞转染自杀基因后,在药物启动转染细胞自杀后,转染细胞及周围未转染细胞均被杀灭。近年来,有人发现局部氧化损伤可以通过细胞间缝隙连接通讯(GJIC)在上皮细胞间传递。以上所述存在着一个共同的现象,就是某些有害因素引起的靶细胞损伤不仅仅局限于靶细胞本身,还可以引起周围正常细胞产生损伤。暴露于有害因素的细胞群会h出现明显的生物效应,这种效应在暴露细胞群和周围未暴露细胞群都会发生,这一现象人们称之为旁观者效应(bystander effect,BE)。旁观者效应是指正常细胞在直接受到有害因素损害的靶细胞诱导下发生的诸如细胞死亡、基因突变、染色体不稳定等反应,这些经诱发而发生效应的正常细胞成为旁观者细胞。由于旁观者效应的存在,使靶细胞的损伤效应得以延续与放大,从而使损伤明显加重。这些研究提醒人们：机体对损伤的反应不仅仅是单个独立细胞或者组织,而是具有群体性。
对于旁观者效应的发现已有几十年,但至今为止,旁观者效应的确切机制尚未阐明。越来越多研究表明,旁观者效应可能与缝隙连接蛋白(connexin, Cx)介导的细胞间隙连接通讯(gap-junctional intercellular communication, GJIC)有密切关系。缝隙连接蛋白是构成细胞间缝隙连接通道基本结构和功能的一类膜蛋白。由六个穿膜的缝隙连接蛋白组成中空的亲水性六聚体结构叫做连接小体(connexon)或缝隙连接半通道(gap junction hemichannel)。在正常情况下,细胞膜对应面上的一对连接小体组成了GJ通道,它能允许分子量低于1KD或直径小于1.5nm的小分子物质如代谢分子、离子、第二信使(如Ca2+、ATP)等通过,这些小分子参与细胞间物质交换的代谢藕联和电信号传递的电藕联,进行细胞间信息传递,从而在细胞的分化、生长、生理功能的调节中发挥重要作用。
铅毒物可增加细胞内活性氧簇(ROS)的水平,同时减少抗氧化物质的浓度活性,破坏氧化、抗氧化平衡,导致氧化损伤,诱发细胞凋亡。同时铅还可通过影响一些相关基因的表达,影响细胞凋亡。目前铅诱导细胞凋亡机理的研究已经取得了许多进展,但尚不完善,铅中毒是否会发生旁观者效应未见报道,铅中毒的毒性代谢产物能否通过间隙连接细胞间通讯(GJIC)进行传导等问题仍有待于深入研究。
综上所述,我们选择神经元模式细胞-大鼠肾上腺嗜铬细胞瘤细胞(PCl2)为研究对象,希望通过对染毒细胞与未染毒细胞共培养体系的研究,对醋酸铅诱导PC12细胞旁观者效应的过程有一个系统、完整的认识,同时为防治铅中毒的下一步研究提供依据。
研究目的：
1、利用直接接触混合共培养技术,构建可以区分醋酸铅染毒与未染毒PC12细胞的共培养体系；
2、通过混合共培养体系,观察醋酸铅染毒PC12细胞对周边正常PC12细胞的影响；
3、研究缝隙连接蛋白介导的细胞间缝隙连接是否参与醋酸铅诱导的旁观者效应,旨在探讨醋酸铅诱导PC12细胞旁观者效应可能的毒理机制。
研究方法：
1、确定醋酸铅的细胞毒性并筛选适合诱导PC12旁观者效应的醋酸铅浓度
(1)细胞增殖能力检测
按照醋酸铅浓度将细胞分成5个实验组(0、0.01、0.1、1、10、100μM),通过MTT比色法,检测不同浓度醋酸铅对PC12细胞增殖能力的影响。分别以每孔5x103个细胞接种到96孔板,暴露于不同浓度的醋酸铅,每孔加MTI镕液20ul,继续培养24h后终止培养,弃培养上清液,每孔加150ul二甲基亚砜(DMSO),置摇床上低速振荡10min,使结晶物充分融解,选择490nm波长,通过酶联免疫监测仪测定各孔光吸收值。
(2)ROS水平检测
将PC12细胞分成2个实验组：PC12、PC12(10μM Pb处理)。各组细胞孵育24h融合后进行实验,弃培养液,室温下D-Hank's漂洗3次,加入含有10μMDHE荧光探针无血清DMEM培养液1mL,37℃避光孵育20min,用无血清培养液洗涤细胞3次,0.25%胰蛋白酶消化,加入1mL无血清培养液吹打均匀,染色完毕后进行Hoechst33342染色。通过荧光显微镜,观察细胞内ROS水平检测。激发波长480～535nm,最大发射波长590nm～610nm; Hoechst33342的最大激发波长为350nm,最大发射波长为460nm。
(3)凋亡相关蛋白检测
按照醋酸铅浓度将细胞分成5个实验组(0、0.01、0.1、1、10、100μM),通过RT-PCR,检测凋亡相关基因(Bax, Bcl-2)的mRNA表达水平,以β-actin做内参照。根据NCBI所提供的基因序列,通过引物设计软件Primer3设计相应引物,扩增条件为预变性：94℃for5min;变性94℃30s,退火60℃30s,延伸72℃1mmin,30个循环；后延伸72℃5min。
(4)细胞凋亡检测
按照醋酸铅浓度将细胞分成5个实验组(0,0.01,0.1,1,10,100μM),通过Annexin v-PE/7-AAD试剂盒,检测PC12细胞凋亡情况。收集1～5×105细胞；加入500μL的Binding Buffe悬浮细胞；加入1μL Annexin V-PE混匀；室温、避光、反应5～15min；加入5μL7-AAD染液,混匀；室温、避光、反应5-15min；在1h内完成流式细胞仪的检测。
(5)线粒体跨膜电位
将细胞分成3个实验组：PC12、GFP-PC12、GFP-PC12(10μM Pb处理)将各组细胞培养24h后,分别加入20nM TMRM,通过流式细胞仪检测线粒体跨膜电位。
(6)NAC处理
将细胞分成3个实验组：PC12、PC12+10μM Pb、PC12(NAC处理)+10μMPb,分别检测各组细胞ROS、线粒体膜电位、凋亡水平(方法同上)。
2、构建染毒细胞与正常细胞共培养体系,并观察检测醋酸铅诱导PC12旁观者效应
(1)构建EF1A-eGFP稳定转染PC12细胞株
将处于对数生长期的PC12细胞吸去培养液,用PBS洗涤后加入适量Trypsin-EDTA,消化后移至离心管中,取1×105cells/9.6cm2的密度接种至六孔板,待细胞完全贴壁后,即可进行转导。转导前,每孔细胞换成1.5mL新鲜的HD+10%FBS培养基或1640+20%FBS培养基,每株各选取一孔细胞加入30μL的Lenti-eGFP/Neo,充分的摇匀后放入37℃、5%CO2、95%相对湿度的培养箱中培养。转导7小时后,吸去含有Lenti-eGFP/Neo的完全培养基,换成新鲜的HD+10%FBS培养基或1640+20%FBS培养基,放入37℃、5%CO2、95%相对湿度的培养箱中培养。转导48小时后,于荧光显微镜下观察转导效果。
(2)细胞混合培养荧光显微镜观测
将PC12、GFP-PC12按比例混合培养,用PBS制备50μM Hoechst33342染料,在37℃培养细胞20分钟,用PBS冲洗细胞两次,用带有350nm激发波长,460nm发射波长的滤光片的荧光显微镜观察细胞。
(3)共培养体系ROS水平、凋亡水平、线粒体跨膜电位检测：
将细胞分成2个实验组组：GFP-PC12+PC12、GFP-PC12(10μMPb处理)+PC12。分别进行ROS水平、凋亡水平、线粒体跨膜电位检测(方法同上)。
3、GJIC在醋酸铅诱导PC12旁观者效应中的作用研究
(1)共培养体系缝隙连接功能检测
将细胞分成2个实验组：GFP-PC12(10μMMPb处理)+PC12、GFP-PC12(10μMPb处理)+PC12+CBX;通过细胞接种荧光示踪法(Parachute Assay)检测细胞间荧光传递功能,即细胞GJ功能。
(2) GJIC阻断剂甘珀酸(CBX)对旁观者效应的影响
将细胞分成2个实验组:GFP-PC12+PC12、GFP-PC12(10μMPb处理)+PC12、GFP-PC12(10μMPb处理)-PC12+CBX。分别进行ROS、凋亡、线粒体跨膜电位检测(方法同上)。
4、数据处理
上述实验至少重复三次。各组间的检验如方差齐则采用单因素方差分析,两两比较采用LSD方法；如方差不齐则采用Welch方法,两两比较采用Dunnett-T3方法,实验结果以平均值±标准差表示。全部实验数据采用SPSS19.0统计软件进行分析,p<0.05视为差异具有统计学意义。
主要结果：
1、醋酸铅引起PC12细胞增殖能力显著下降,凋亡水平上升。
MTT法检测结果显示各剂量组细胞活性之间有显著性差异(F值=386.421,P=0.000),其中暴露于1μm醋酸铅的PC12细胞增殖能力降低(95.0%±3.8%),与对照组比较有显著性差异(P=0.04),而暴露于10μM和100μm醋酸铅的PC12细胞增殖能力分别为69.8%±2.8%和51.4%±3.8%(P=0.00；P=0.00)。流式细胞仪结果显示,0.1μM、1μM、10μM, and100μM暴露组的凋亡率分别为1.33%±0.42%,5.40%±0.51%,38.33%±3.06%,46.67%±4.51%。说明随着醋酸铅染毒浓度的升高,细胞的增殖能力逐渐下降,凋亡水平逐步升高。
2、醋酸铅引起PC12细胞凋亡相关基因Bax表达上升,Bcl-2表达下降,Bax/Bcl-2比值上升,并呈剂量反应关系。
RT-PCR分析结果显示,各剂量组细胞Bax基因mRNA表达之间有显著性差异(Welcl值=103.674,P=0.000),其中1μM、10μM、100μM组细胞Bax基因mRNA表达与对照组细胞比较有显著性差异(P=0.011；P=0.034；P=0.008),说明随着醋酸铅浓度增高,细胞Bax基因mRNA表达逐渐下降。各剂量组细胞Bcl-2基因mRN表达之间有显著性差异(Welch值=363.691,P=0.000),其中10μ M、100μm组细胞Bcl-2基因,mRJA表达与对照组细胞比较有显著性差异(P=0.049；P=0.027),说明随着醋酸铅浓度增高,细胞Bcl-2基因mRNA表达逐渐升高。
3、抗氧化剂N-乙酰基半胱氨酸(NAC)可以抑制醋酸铅引起的ROS水平增高、线粒体跨膜电位降低及细胞凋亡。
经过NAC预处理后,荧光显微镜下,NAC预处理组ROS生成水平明显低于未处理组(P=0.000)；线粒体跨膜电位NAC预处理组高于未处理组(P=0.000)；暴露10μM醋酸铅的PC12细胞凋亡率下降至14.33%±1.53%。
4、通过EF1A-EGFP稳定转染PC12细胞,构建了共培养体系,在荧光显微镜下可以区分染毒与未染毒细胞。
荧光显微镜下,稳定转染PC12细胞呈现绿色加蓝色核染,而正常细胞仅为蓝色核染。
5、细胞荧光免疫示踪法(Parachute Assay)显示GFP-PC12(Pb2+)细胞和PC12细胞之间建立了细胞间隙连接通讯。
荧光显微镜下稳定转染PC12细胞将绿色染料传递给周围正常细胞,CBX可以有效组织这种传递。说明稳定转染PC12细胞与正常细胞之间建立了缝隙连接。
6、GJIC阻断剂甘珀酸(CBX)可以部分阻断醋酸铅诱发的旁观者效应
在共培养体系中,ROS水平GJIC阻断剂甘珀酸(CBX)处理前PC12细胞的(3.48±0.59),处理后为(1.64±0.42),差异有显著性(P=0.000)。凋亡水平GJIC阻断剂甘珀酸(CBX)处理前为(43.52%±8.07%),处理后为(12.19%±1.13%),差异有显著性(P=0.000)。线粒体跨膜电位处理前低于处理后。
结论：
1、醋酸铅可诱导PC12细胞凋亡的线粒体途径。
2、醋酸铅对PC12细胞造成的损伤可以传递给周围正常细胞。
3、细胞间隙连接通讯GJIC在铅诱导的旁观者效应中起了重要的作用。
Background
Pb is one of the most commonly used metals in industry and is highly toxic. Pb can enter the human body mainly through inhalation and gastrointestinal absorption. In cells, Pb can increase the intracellular reactive oxygen species (ROS) levels and reduce the concentration and the activity of antioxidant enzymes, which disturbs the oxidative-antioxidative balance and induces apoptosis. High levels of Pb can cause irreversible damage to the nervous system. However, it was recently discovered that blood Pb at acceptable levels may impact the intellectual development of children. Thus, the neurotoxicity of Pb attracts considerable attention.
It is known that cells can influence each other in a co-culture microenvironment. Over the past two decades, classical radiation biology research, which focuses on the irradiated cells themselves as the main target of radiation-induced damage, had been challenged by a significant amount of scientific evidence that clearly shows that radiation-induced bystander signals play important roles in mediating the overall radiobiological response. The radiation-induced bystander effects refer to those responses that occur in cells that were not subject to energy deposition events following ionizing radiation. The bystander effect is characterized by a signaling process from irradiated cells to non-irradiated cells that causes oxidative damage in neighboring cells. Bystander effects have been observed in both in vitro and in vivo toxicological studies. It been proposed that the bystander effect of gene therapy, when the part of the tumor cells transfected with a suicide gene, after start the drug suicide, the transfected cells and the surrounding non-transfected cells were killed.
To date, the mechanisms underlying these bystander effects have not been well studied. This mechanism may be related to the active signaling molecule transferred between cells via gap junctions. Gap-junctions, which are composed of six transmembrane connexin subunits arranged as cylindrical channels (-1.5-nm diameter) between connecting adjacent cells, can facilitate the transfer of1-to3-kDa molecules with some dependence on the cell type and physiological status. The involvement of this route of communication among neighboring cells was also demonstrated in neuronal and myocardial ischemia-reperfusion injuries, as well as in liver injuries. Accordingly, it is reasonable to speculate on the damage that occurs not only on the target cells but also on the neighboring cells, and researchers have hypothesized that gap-junctional intercellular communication (GJIC) plays an important role in mediating the bystander effect.
Thus, it is unclear whether acceptable blood Pb levels impact the intellectual development by themselves and whether the injured cells transmit the damaged information to neighboring cells. Although Pb-induced apoptosis is a well-reported phenomenon, studies of the bystander effect and the underlying cellular mechanisms are lacking. In this study, we developed a novel co-culture method in which PC12cells were cultured with direct cell-to-cell contact with GFP-labeled PC12(GFP-PC12) cells pretreated with lead acetate. The apoptosis, intracellular ROS generation, and mitochondrial membrane potential (MMP) of PC12cells were subsequently detected. A ROS scavenger and gap junction blocker were used to explore the hypothesis that the Pb-induced oxidative damage can propagate via GJIC.
Methods:
1. MTT assay
The cell viability was determined through the MTT assay. Approximately5x103cells were plated in each well of a96-well plate for24h, and the PC12cells were then exposed to0-100μM lead acetate for24h. Subsequently, the medium was removed, and20μl of5mg/ml MTT (dissolved in PBS) and18μl of DMEM were added to each well. The plate was then incubated at37℃for4h. The medium was removed, and the purple formazan crystals were dissolved in150μl of DMSO. The absorbance of each well was measured at490nm (Power Wave XS; Bio-Tek, Winooski, VT, USA). All of the samples were assayed in six duplicates.
2. RNA extraction and reverse transcription polymerase chain reaction (RT-PCR)
Lead acetate can induce cell apoptosis. To determine the exact concentration of lead acetate required for the induction of apoptosis, the expression levels of the Bax and Bcl-2genes, which are the important apoptosis-related genes, were detected by RT-PCR. The total RNA was extracted according to the manufacturer's instructions and was reverse-transcribed using Trizol reagents. The primer exhibited as Table1. The PCR was performed with the following temperature program:denaturation at94℃for5min followed by30cycles of denaturation at94℃for30sec, annealing at60℃for30s, and extension at72℃for5min (n=3). The PCR reaction products were detected through gel electrophoresis and ultraviolet transillumination.
3. Flow cytometry analyses
3.1. Apoptosis and cell death
After induction with lead acetate and co-culture, the cells were rinsed twice with cold PBS and resuspended in binding buffer, and the degree of apoptosis and cell death were then assessed by flow cytometry using an Annexin V-PE/7-AAD kit according to the manufacturer's instructions. The final suspension was analyzed using a FACS Calibur flow cytometer (BD Biosciences), and the results were analyzed using the CellQuest Pro software (BD Biosciences).
3.2. Measurements of mitochondrial membrane potential (△ψm)
The△ψm, which has been suggested to be central to the apoptotic pathway (Ly et al.,2003), was measured by flow cytometry using the TMRM detection kit according to the manufacturer's instructions. PC12cells were incubated in the presence of50nM TMRM for30min at room temperature in the dark and then stored on ice until analysis by flow cytometry. For each sample,10,000particles were analyzed. The final suspension was analyzed using a FACS Calibur flow cytometer, and the results were analyzed using the CellQuest Pro software. The arithmetic mean values of the fluorescence signals in arbitrary units were determined for each sample and graphically represented. All of the experiments were repeated three times.
4. ROS detection
Dihydroethidium (DHE) was used to measure the ROS generation in cells as previously reported with slight modifications. Briefly, at the end of the treatment period, the cells were washed and incubated with2.5μM DHE (red) at37℃for30min and Hoechst33342(blue) to stain the nucleus for30min. Fluorescence microscopy was used to capture the differences in the red fluorescence intensities. The examination was performed with a laser-scanning confocal microscope (LSM510META; Carl Zeiss, Hamburg, Germany). The images were analyzed with the Image-Pro Plus software (version6.0). The mean fluorescence intensity of each cell was calculated, and the total cell emission signals per field were averaged for data analysis.
5. Stable transfection of PC12cells with EF1A-eGFP.
To distinguish the normal PC12cells from the lead-induced PC12cells in the direct cell-to-cell contact system, some of the PC12cells were transfected with the EF1A-eGFP vector (GFP-PC12) and selected with400μg/ml G418. The transfections were performed according to the standard protocol from Cyagen. The expression of GFP was identified by fluorescence microscopy and flow cytometry analysis.
6. Co-culture system to determine the bystander effects of lead-induced PC12cells
6.1Lead acetate-induced PC12-GFP cells co-cultured with PC12-GFP (Pb2+) cells
The GFP-PC12cells were exposed to different concentrations (0.1μM,1μM,10μM, and100μM) of lead acetate (dissolved in pure water) after incubation for24h. The exposures were performed in normal growth medium. The control cells were treated with5mM NAC, which is commonly used to identify and inhibit ROS, for1h and then exposed to10μM lead acetate for24h (GFP-PC12+NAC+Pb). The cells were harvested with0.025%trypsin and0.02%EDTA (dissolved in PBS and degermed).
6.2The bystander effects of PC12-GFP (Pb2+) cells is determined by direct cell to cell contact system.
The GFP-PC12cells (1×103cells/ml) were co-cultured with PC12cells (5×103cells/ml) in three groups. First, GFP-PC12cells co-cultured with PC12cells were assumed to be the control group (GFP-PC12+PC12). GFP-PC12(Pb2+) cells co-cultured with PC12cells (GFP-PC12(Pb2+)+PC12) were used to determine the bystander effects of the induced cells. To determine the effect of CBX, an inhibitor of gap junction, on the bystander effect, GFP-PC12(Pb2+) cells were co-cultured with PC12cells in medium with100μM CBX (GFP-PC12(Pb2+)+PC12+CBX), The cells were harvested after24h of co-culture.
7. Parachute assay for gap junction function
To confirm the function of gap junctions, which was hypothesized to underline the bystander effect, in a co-culture system, the parachute assay was used. To avoid overlaps between the green fluorescences of GFP and calcein-AM, which is converted intracellularly into the gap junction-permeable dye calcein. Briefly, before co-culture, GFP-PC12(Pb2+) cells were double-labeled with5μM CM-Dil, a membrane dye that does not spread to coupled cells, and5μM calcein-acetoxymethyl ester, which is converted intracellularly into the gap junction-permeable dye calcein. After incubation for30min, the GFP-PC12(Pb2+) cells were then trypsinized, diluted to500cells/ml, and used as the donor cells. Two groups of PC12cells were plated in12-well plates as receptor cells:one group of cells was incubated with1ml of the donor solution, and the other group of cells was incubated with1ml of the donor solution supplemented with100μM CBX, which inhibits the formation of gap junctions without affecting the activity of connexin. The cells were incubated for approximately4h in5%CO2at37℃and then examined with an inverted fluorescence microscope.
8. Statistic
The above test needs to be performed at least three times. The results for the between-group tests are presented by "mean±standard deviation". The between-group tests are divided into two cases according to the type of variance. When homogeneity of variance holds, we use one-way analysis of variance and LSD method forpairwise comparison. When heterogeneity of variance holds, we use Welch method and Dunnett-T3for pairwise comparison. And the results for the between-group tests are presented "mean±standard deviation". We use SPSS19.0to analyze the data. The difference is statistically significant if "P<0.05".
The main results:
1. Lead acetate is toxic to PC12cells dose-dependentty. The toxic can be inhibited by NAC partly
After the incubation of PC12cells with different concentrations of lead acetate (0.1-100μM) for24h, the MTT assay, flow cytometry, and RT-PCR were used to detect the effects of lead acetate on PC12cells.
1.1Lead acetate is toxic to the viability of PC12cell dose-dependenlly
The cell viability was determined by the MTT assay. The viability of the control cells was normalized to1, and the viabilities of the other cells are expressed as percentages compared with that of the control. The exposure to1μM of lead acetate significantly (0.01 1.2Effect of lead acetate on Bax and Bcl-2levels
To determine the exact concentration of lead acetate for use in the subsequent studies, we performed RT-PCR to detect the gene expression levels of Bax and Bcl-2in PC12cells after treatment with lead acetate at different concentrations. All of the RT-PCR results were standardized to the levels observed in normal PC12cells (normalized to1).The RT-PCR analysis demonstrated that the expression of Bax mRNA was significantly (P<0.01,n=3) increased to3.55,3.18, and4.42after treatment with1μM,10μM, and100μM lead acetate, respectively, compared with the untreated PC12cells; however, the0.1μM group did not exhibit a significant difference. However, the differences between these treatment groups were not significant. The expression levels of Bcl-2mRNA were decreased significantly in the10μM and100μM groups (33.3%±0.55%and4.42%±0.14%, respectively, P<0.01, n=3). The analysis of the express ion levels of Bax and Bcl-2mRNA also suggests that a lead concentration of10μM is sufficient to induce cytotoxic effects in PC12cells.
1.3Lead acetate is toxic to the apoptosis rate of PC12cells dose-dependently. NAC can inhibit the apoptosis partly.
Tthe flow cytometry analysis revealed that the percentage of apoptosis in the untreated PC12cell population was0.40%±0.46%, and significantly higher percentages of PC12cells underwent apoptosis after treatment with0.1μM,1μM,10μM, and100μM(1.33%±0.42%,5.40%±0.51%,38.33%±3.06%, and,46.67%±4.51%, respectively). The percentages of apoptotic cells in the10μM and100μM groups were not significantly different. However, a further study showed that18.67%±2.08%of the cells in the100μM group died, whereas a lower percentage of cells (9.97%±1.95%) died in the10μM group (P<0.01, n=3). Consistent with the MTT and RT-PCR results, a lead acetate concentration of10μM was selected for the subsequent studies. NAC, a commonly used inhibitor of ROS generation, was used to determine the probable mechanism underlying lead-induced apoptosis. The data showed that pre-treatment with5mM NAC decreased the percentage of apoptotic cells to14.33%±1.53%and the percentage of dead cells was to5.13%±0.81%. This result indicates that NAC partly inhibits the apoptosis induced by lead acetate.
1.4Effect of lead acetate on ROS generation
The immunofluorescent staining shown that less PC12cells (Hoechst33342, blue) express ROS (red), and the cells exhibit a low fluorescence prior to exposure to lead acetate. After treatment with10μM lead acetate for24h, the ROS generation increased markedly. It is interesting that pre-treatment with5nM NAC for30min can inhibit ROS generation in PC12cells that are exposed to lead acetate.
To distinguish the exposed cells and unexposed PC12cells, the former were successfully and stably transfected with the EF1A-EGFP vector (GFP-PC12) before induced, and approximately99%of the GFP-PC12cells were found to be GFP-positive, as evaluated using an inverted fluorescence microscope and flow cytometry.
To exclude the effect of transfected GFP on ROS generation in PC12cells, fluorescence microscopy was used to capture the fluorescence intensities associated with ROS generation in PC12cells after transfection with GFP. Compared with normal PC12cells (normalized to1), the ROS generation levels in GFP-PC12cells, GFP-PC12+Pb cells, and GFP-PC12+NAC+Pb cells were0.92±0.15,3.97±0.47and1.09±0.16, respectively. These data show that transfected GFP has no effect on ROS generation and that GFP-PC12+Pb cells produced a significantly higher amount of ROS. Pre-treatment with NAC can inhibit the subsequent ROS generation even if the cells are incubated with lead acetate at a later point in time.
1.5NAC can inhibit the toxic of lead acetate on mitochondrial membrane potential (△ψm) of PC12cells dose-dependently mainly
Fluorescent probes for monitoring△ψm are frequently used for the assessment of mitochondrial function, particularly in the context of oxidative stress and apoptosis research. The△ψm of PC12cells, GFP-PC12cells, GFP-PC12+Pb cells, and GFP-PC12+NAC+Pb cells was determined through FACS analysis using the fluorescent probe TMRM (red). The GFP-PC12+Pb cells expressed the lowest Aym, and the cells in the other groups showed the same intensity compared with the PC12group.
2. The bystander effects of GFP-PC12(Pb24) cells are mediated by GJIC and inhibited by CBX
After successful transfection, the GFP-PC12cells were incubated in normal medium containing10μM lead acetate for24h, and the GFP-PC12(Pb2+) cells were then co-cultured with normal PC12cells.
2.1CBX inhibits functional GJIC in the co-culture system
The parachute assay results demonstrate that functional gap junctions were formed between GFP-PC12(Pb2+) cells and PC12cells. The red fluorescence represents the donor cells, e.g., GFP-PC12(Pb2+) cells, and the green fluorescence demonstrated that the donor cells transmitted the signals to the receptor cells (PC12cells) through functional gap junctions. After incubation with CBX, an inhibitor of functional gap junctions, the donor cells did not convey any signals.
2.2The ROS generation in PC12cells in the co-culture system can be inhibited by CBX
Images of the immunofluorescent staining and the diagram shown indicates that minimal red fluorescence was detected in the GFP-PC12+PC12cells (normalized to1). However, almost all of the GFP-PC12(Pb2+)+PC12cells expressed a significantly higher level of red fluorescence in the nucleus (3.48±0.59). In contrast, pre-treatment with CBX resulted in a lower level of red fluorescence (1.64±0.42) compared with that expressed by the GFP-PC12(Pb2+)+PC12cells (P<0.01, n=3) and a higher level of fluorescence than that observed in the GFP-PC12+PC12cells (P<0.01,n=3).
2.3The apoptosis of PC12cells in the co-culture system can be inhibited by CBX
In the co-culture system, GFP-PC12(Pb2+) cells were GFP(+), whereas the co-cultured PC12cells were GFP(-). To detect the apoptosis of the co-cukured PC12cells, the Annexin V-PE/7-AAD apoptosis detection kit, which stains apoptosis cells a red color, was used. The Annexin V(+)GFP(-) cells represent cells that were affected by the bystander effect and thus need to be further analyzed.The Annexin V(+)GFP(-) PC12cells in the co-culture system endure the highest percentage of apoptosis (43.52%±8.07%). CBX significantly inhibits cell apoptosis (12.19%±1.13%).
2.4The changes in the mitochondrial membrane potential (△ψm) of PC12cells in the co-culture system can be inhibited by CBX
A FACS analysis was used to measure the△ψm in GFP-PC12+PC12, GFP-PC12(Pb24)+Pb, and GFP-PC12(Pb2+)+Pb+CBX cells using the fluorescent probe TMRM. In the co-culture system, only the TMRM(+)GFP(-) cells were investigated. The selected cells from the GFP-PC12(Pb2+)+Pb group exhibited the lowest△ψm, and the cells in the GFP-PC12(Pb2+)+Pb+CBX group presented a lower intensity compared with the GFP-PC12+PC12group. This finding demonstrates that CBX can inhibit the lead-induced changes in△ψm.
Conclusions:
1. Lead acetate can induce the mitochondrial pathway of apoptosis in PC12cells.
2. In co-culture system, Pb-exposed GFP-PC12cells exerted bystander effects that resulted in increased apoptosis, the generation of ROS, and the collapse of the MMP of the untreated PC12cells.
3. The bystander effects, which drive the expansion of the Pb-induced damage to neighboring cells, were determined to be dependent on functional GJIC.
铅是工业上常用的重金属之一,可以通过呼吸和消化系统进入人体。铅具有很强的神经毒性,可导致不可逆的神经系统损伤,儿童在发育期对铅暴露产生的毒性尤为敏感。科学家们发现即使儿童的血铅浓度保持在可接受水平,儿童的智力发育仍会受到明显影响。因此,铅的神经毒性受到了广泛关注。
细胞生长的微环境是保持细胞正常增殖、分化、代谢和功能活动的重要条件。细胞对外界的微环境非常敏感,在同一共培养体系中,细胞之间或细胞与胞外基质之间的直接接触可以影响细胞的生长状态。近几十年来,在放射生物学领域发现,当少数细胞受到射线照射时,没有受到射线直接照射的周围细胞也能表现出一些损伤效应。大量研究已经证明在体内和体外实验中确实存在损伤传递效应。例如,慢性粒细胞白血病患儿的脾部在接受放射线照射治疗时,胸骨骨髓细胞发生了损伤；局部氧化损伤可以通过GJIC在内皮细胞之间传递。有人提出用肿瘤自杀基因治疗肿瘤,当部分肿瘤细胞转染自杀基因后,在药物启动转染细胞自杀后,转染细胞及周围未转染细胞均被杀灭。近年来,有人发现局部氧化损伤可以通过细胞间缝隙连接通讯(GJIC)在上皮细胞间传递。以上所述存在着一个共同的现象,就是某些有害因素引起的靶细胞损伤不仅仅局限于靶细胞本身,还可以引起周围正常细胞产生损伤。暴露于有害因素的细胞群会h出现明显的生物效应,这种效应在暴露细胞群和周围未暴露细胞群都会发生,这一现象人们称之为旁观者效应(bystander effect,BE)。旁观者效应是指正常细胞在直接受到有害因素损害的靶细胞诱导下发生的诸如细胞死亡、基因突变、染色体不稳定等反应,这些经诱发而发生效应的正常细胞成为旁观者细胞。由于旁观者效应的存在,使靶细胞的损伤效应得以延续与放大,从而使损伤明显加重。这些研究提醒人们：机体对损伤的反应不仅仅是单个独立细胞或者组织,而是具有群体性。
对于旁观者效应的发现已有几十年,但至今为止,旁观者效应的确切机制尚未阐明。越来越多研究表明,旁观者效应可能与缝隙连接蛋白(connexin, Cx)介导的细胞间隙连接通讯(gap-junctional intercellular communication, GJIC)有密切关系。缝隙连接蛋白是构成细胞间缝隙连接通道基本结构和功能的一类膜蛋白。由六个穿膜的缝隙连接蛋白组成中空的亲水性六聚体结构叫做连接小体(connexon)或缝隙连接半通道(gap junction hemichannel)。在正常情况下,细胞膜对应面上的一对连接小体组成了GJ通道,它能允许分子量低于1KD或直径小于1.5nm的小分子物质如代谢分子、离子、第二信使(如Ca2+、ATP)等通过,这些小分子参与细胞间物质交换的代谢藕联和电信号传递的电藕联,进行细胞间信息传递,从而在细胞的分化、生长、生理功能的调节中发挥重要作用。
铅毒物可增加细胞内活性氧簇(ROS)的水平,同时减少抗氧化物质的浓度活性,破坏氧化、抗氧化平衡,导致氧化损伤,诱发细胞凋亡。同时铅还可通过影响一些相关基因的表达,影响细胞凋亡。目前铅诱导细胞凋亡机理的研究已经取得了许多进展,但尚不完善,铅中毒是否会发生旁观者效应未见报道,铅中毒的毒性代谢产物能否通过间隙连接细胞间通讯(GJIC)进行传导等问题仍有待于深入研究。
综上所述,我们选择神经元模式细胞-大鼠肾上腺嗜铬细胞瘤细胞(PCl2)为研究对象,希望通过对染毒细胞与未染毒细胞共培养体系的研究,对醋酸铅诱导PC12细胞旁观者效应的过程有一个系统、完整的认识,同时为防治铅中毒的下一步研究提供依据。
研究目的：
1、利用直接接触混合共培养技术,构建可以区分醋酸铅染毒与未染毒PC12细胞的共培养体系；
2、通过混合共培养体系,观察醋酸铅染毒PC12细胞对周边正常PC12细胞的影响；
3、研究缝隙连接蛋白介导的细胞间缝隙连接是否参与醋酸铅诱导的旁观者效应,旨在探讨醋酸铅诱导PC12细胞旁观者效应可能的毒理机制。
研究方法：
1、确定醋酸铅的细胞毒性并筛选适合诱导PC12旁观者效应的醋酸铅浓度
(1)细胞增殖能力检测
按照醋酸铅浓度将细胞分成5个实验组(0、0.01、0.1、1、10、100μM),通过MTT比色法,检测不同浓度醋酸铅对PC12细胞增殖能力的影响。分别以每孔5x103个细胞接种到96孔板,暴露于不同浓度的醋酸铅,每孔加MTI镕液20ul,继续培养24h后终止培养,弃培养上清液,每孔加150ul二甲基亚砜(DMSO),置摇床上低速振荡10min,使结晶物充分融解,选择490nm波长,通过酶联免疫监测仪测定各孔光吸收值。
(2)ROS水平检测
将PC12细胞分成2个实验组：PC12、PC12(10μM Pb处理)。各组细胞孵育24h融合后进行实验,弃培养液,室温下D-Hank's漂洗3次,加入含有10μMDHE荧光探针无血清DMEM培养液1mL,37℃避光孵育20min,用无血清培养液洗涤细胞3次,0.25%胰蛋白酶消化,加入1mL无血清培养液吹打均匀,染色完毕后进行Hoechst33342染色。通过荧光显微镜,观察细胞内ROS水平检测。激发波长480～535nm,最大发射波长590nm～610nm; Hoechst33342的最大激发波长为350nm,最大发射波长为460nm。
(3)凋亡相关蛋白检测
按照醋酸铅浓度将细胞分成5个实验组(0、0.01、0.1、1、10、100μM),通过RT-PCR,检测凋亡相关基因(Bax, Bcl-2)的mRNA表达水平,以β-actin做内参照。根据NCBI所提供的基因序列,通过引物设计软件Primer3设计相应引物,扩增条件为预变性：94℃for5min;变性94℃30s,退火60℃30s,延伸72℃1mmin,30个循环；后延伸72℃5min。
(4)细胞凋亡检测
按照醋酸铅浓度将细胞分成5个实验组(0,0.01,0.1,1,10,100μM),通过Annexin v-PE/7-AAD试剂盒,检测PC12细胞凋亡情况。收集1～5×105细胞；加入500μL的Binding Buffe悬浮细胞；加入1μL Annexin V-PE混匀；室温、避光、反应5～15min；加入5μL7-AAD染液,混匀；室温、避光、反应5-15min；在1h内完成流式细胞仪的检测。
(5)线粒体跨膜电位
将细胞分成3个实验组：PC12、GFP-PC12、GFP-PC12(10μM Pb处理)将各组细胞培养24h后,分别加入20nM TMRM,通过流式细胞仪检测线粒体跨膜电位。
(6)NAC处理
将细胞分成3个实验组：PC12、PC12+10μM Pb、PC12(NAC处理)+10μMPb,分别检测各组细胞ROS、线粒体膜电位、凋亡水平(方法同上)。
2、构建染毒细胞与正常细胞共培养体系,并观察检测醋酸铅诱导PC12旁观者效应
(1)构建EF1A-eGFP稳定转染PC12细胞株
将处于对数生长期的PC12细胞吸去培养液,用PBS洗涤后加入适量Trypsin-EDTA,消化后移至离心管中,取1×105cells/9.6cm2的密度接种至六孔板,待细胞完全贴壁后,即可进行转导。转导前,每孔细胞换成1.5mL新鲜的HD+10%FBS培养基或1640+20%FBS培养基,每株各选取一孔细胞加入30μL的Lenti-eGFP/Neo,充分的摇匀后放入37℃、5%CO2、95%相对湿度的培养箱中培养。转导7小时后,吸去含有Lenti-eGFP/Neo的完全培养基,换成新鲜的HD+10%FBS培养基或1640+20%FBS培养基,放入37℃、5%CO2、95%相对湿度的培养箱中培养。转导48小时后,于荧光显微镜下观察转导效果。
(2)细胞混合培养荧光显微镜观测
将PC12、GFP-PC12按比例混合培养,用PBS制备50μM Hoechst33342染料,在37℃培养细胞20分钟,用PBS冲洗细胞两次,用带有350nm激发波长,460nm发射波长的滤光片的荧光显微镜观察细胞。
(3)共培养体系ROS水平、凋亡水平、线粒体跨膜电位检测：
将细胞分成2个实验组组：GFP-PC12+PC12、GFP-PC12(10μMPb处理)+PC12。分别进行ROS水平、凋亡水平、线粒体跨膜电位检测(方法同上)。
3、GJIC在醋酸铅诱导PC12旁观者效应中的作用研究
(1)共培养体系缝隙连接功能检测
将细胞分成2个实验组：GFP-PC12(10μMMPb处理)+PC12、GFP-PC12(10μMPb处理)+PC12+CBX;通过细胞接种荧光示踪法(Parachute Assay)检测细胞间荧光传递功能,即细胞GJ功能。
(2) GJIC阻断剂甘珀酸(CBX)对旁观者效应的影响
将细胞分成2个实验组:GFP-PC12+PC12、GFP-PC12(10μMPb处理)+PC12、GFP-PC12(10μMPb处理)-PC12+CBX。分别进行ROS、凋亡、线粒体跨膜电位检测(方法同上)。
4、数据处理
上述实验至少重复三次。各组间的检验如方差齐则采用单因素方差分析,两两比较采用LSD方法；如方差不齐则采用Welch方法,两两比较采用Dunnett-T3方法,实验结果以平均值±标准差表示。全部实验数据采用SPSS19.0统计软件进行分析,p<0.05视为差异具有统计学意义。
主要结果：
1、醋酸铅引起PC12细胞增殖能力显著下降,凋亡水平上升。
MTT法检测结果显示各剂量组细胞活性之间有显著性差异(F值=386.421,P=0.000),其中暴露于1μm醋酸铅的PC12细胞增殖能力降低(95.0%±3.8%),与对照组比较有显著性差异(P=0.04),而暴露于10μM和100μm醋酸铅的PC12细胞增殖能力分别为69.8%±2.8%和51.4%±3.8%(P=0.00；P=0.00)。流式细胞仪结果显示,0.1μM、1μM、10μM, and100μM暴露组的凋亡率分别为1.33%±0.42%,5.40%±0.51%,38.33%±3.06%,46.67%±4.51%。说明随着醋酸铅染毒浓度的升高,细胞的增殖能力逐渐下降,凋亡水平逐步升高。
2、醋酸铅引起PC12细胞凋亡相关基因Bax表达上升,Bcl-2表达下降,Bax/Bcl-2比值上升,并呈剂量反应关系。
RT-PCR分析结果显示,各剂量组细胞Bax基因mRNA表达之间有显著性差异(Welcl值=103.674,P=0.000),其中1μM、10μM、100μM组细胞Bax基因mRNA表达与对照组细胞比较有显著性差异(P=0.011；P=0.034；P=0.008),说明随着醋酸铅浓度增高,细胞Bax基因mRNA表达逐渐下降。各剂量组细胞Bcl-2基因mRN表达之间有显著性差异(Welch值=363.691,P=0.000),其中10μ M、100μm组细胞Bcl-2基因,mRJA表达与对照组细胞比较有显著性差异(P=0.049；P=0.027),说明随着醋酸铅浓度增高,细胞Bcl-2基因mRNA表达逐渐升高。
3、抗氧化剂N-乙酰基半胱氨酸(NAC)可以抑制醋酸铅引起的ROS水平增高、线粒体跨膜电位降低及细胞凋亡。
经过NAC预处理后,荧光显微镜下,NAC预处理组ROS生成水平明显低于未处理组(P=0.000)；线粒体跨膜电位NAC预处理组高于未处理组(P=0.000)；暴露10μM醋酸铅的PC12细胞凋亡率下降至14.33%±1.53%。
4、通过EF1A-EGFP稳定转染PC12细胞,构建了共培养体系,在荧光显微镜下可以区分染毒与未染毒细胞。
荧光显微镜下,稳定转染PC12细胞呈现绿色加蓝色核染,而正常细胞仅为蓝色核染。
5、细胞荧光免疫示踪法(Parachute Assay)显示GFP-PC12(Pb2+)细胞和PC12细胞之间建立了细胞间隙连接通讯。
荧光显微镜下稳定转染PC12细胞将绿色染料传递给周围正常细胞,CBX可以有效组织这种传递。说明稳定转染PC12细胞与正常细胞之间建立了缝隙连接。
6、GJIC阻断剂甘珀酸(CBX)可以部分阻断醋酸铅诱发的旁观者效应
在共培养体系中,ROS水平GJIC阻断剂甘珀酸(CBX)处理前PC12细胞的(3.48±0.59),处理后为(1.64±0.42),差异有显著性(P=0.000)。凋亡水平GJIC阻断剂甘珀酸(CBX)处理前为(43.52%±8.07%),处理后为(12.19%±1.13%),差异有显著性(P=0.000)。线粒体跨膜电位处理前低于处理后。
结论：
1、醋酸铅可诱导PC12细胞凋亡的线粒体途径。
2、醋酸铅对PC12细胞造成的损伤可以传递给周围正常细胞。
3、细胞间隙连接通讯GJIC在铅诱导的旁观者效应中起了重要的作用。
Background
Pb is one of the most commonly used metals in industry and is highly toxic. Pb can enter the human body mainly through inhalation and gastrointestinal absorption. In cells, Pb can increase the intracellular reactive oxygen species (ROS) levels and reduce the concentration and the activity of antioxidant enzymes, which disturbs the oxidative-antioxidative balance and induces apoptosis. High levels of Pb can cause irreversible damage to the nervous system. However, it was recently discovered that blood Pb at acceptable levels may impact the intellectual development of children. Thus, the neurotoxicity of Pb attracts considerable attention.
It is known that cells can influence each other in a co-culture microenvironment. Over the past two decades, classical radiation biology research, which focuses on the irradiated cells themselves as the main target of radiation-induced damage, had been challenged by a significant amount of scientific evidence that clearly shows that radiation-induced bystander signals play important roles in mediating the overall radiobiological response. The radiation-induced bystander effects refer to those responses that occur in cells that were not subject to energy deposition events following ionizing radiation. The bystander effect is characterized by a signaling process from irradiated cells to non-irradiated cells that causes oxidative damage in neighboring cells. Bystander effects have been observed in both in vitro and in vivo toxicological studies. It been proposed that the bystander effect of gene therapy, when the part of the tumor cells transfected with a suicide gene, after start the drug suicide, the transfected cells and the surrounding non-transfected cells were killed.
To date, the mechanisms underlying these bystander effects have not been well studied. This mechanism may be related to the active signaling molecule transferred between cells via gap junctions. Gap-junctions, which are composed of six transmembrane connexin subunits arranged as cylindrical channels (-1.5-nm diameter) between connecting adjacent cells, can facilitate the transfer of1-to3-kDa molecules with some dependence on the cell type and physiological status. The involvement of this route of communication among neighboring cells was also demonstrated in neuronal and myocardial ischemia-reperfusion injuries, as well as in liver injuries. Accordingly, it is reasonable to speculate on the damage that occurs not only on the target cells but also on the neighboring cells, and researchers have hypothesized that gap-junctional intercellular communication (GJIC) plays an important role in mediating the bystander effect.
Thus, it is unclear whether acceptable blood Pb levels impact the intellectual development by themselves and whether the injured cells transmit the damaged information to neighboring cells. Although Pb-induced apoptosis is a well-reported phenomenon, studies of the bystander effect and the underlying cellular mechanisms are lacking. In this study, we developed a novel co-culture method in which PC12cells were cultured with direct cell-to-cell contact with GFP-labeled PC12(GFP-PC12) cells pretreated with lead acetate. The apoptosis, intracellular ROS generation, and mitochondrial membrane potential (MMP) of PC12cells were subsequently detected. A ROS scavenger and gap junction blocker were used to explore the hypothesis that the Pb-induced oxidative damage can propagate via GJIC.
Methods:
1. MTT assay
The cell viability was determined through the MTT assay. Approximately5x103cells were plated in each well of a96-well plate for24h, and the PC12cells were then exposed to0-100μM lead acetate for24h. Subsequently, the medium was removed, and20μl of5mg/ml MTT (dissolved in PBS) and18μl of DMEM were added to each well. The plate was then incubated at37℃for4h. The medium was removed, and the purple formazan crystals were dissolved in150μl of DMSO. The absorbance of each well was measured at490nm (Power Wave XS; Bio-Tek, Winooski, VT, USA). All of the samples were assayed in six duplicates.
2. RNA extraction and reverse transcription polymerase chain reaction (RT-PCR)
Lead acetate can induce cell apoptosis. To determine the exact concentration of lead acetate required for the induction of apoptosis, the expression levels of the Bax and Bcl-2genes, which are the important apoptosis-related genes, were detected by RT-PCR. The total RNA was extracted according to the manufacturer's instructions and was reverse-transcribed using Trizol reagents. The primer exhibited as Table1. The PCR was performed with the following temperature program:denaturation at94℃for5min followed by30cycles of denaturation at94℃for30sec, annealing at60℃for30s, and extension at72℃for5min (n=3). The PCR reaction products were detected through gel electrophoresis and ultraviolet transillumination.
3. Flow cytometry analyses
3.1. Apoptosis and cell death
After induction with lead acetate and co-culture, the cells were rinsed twice with cold PBS and resuspended in binding buffer, and the degree of apoptosis and cell death were then assessed by flow cytometry using an Annexin V-PE/7-AAD kit according to the manufacturer's instructions. The final suspension was analyzed using a FACS Calibur flow cytometer (BD Biosciences), and the results were analyzed using the CellQuest Pro software (BD Biosciences).
3.2. Measurements of mitochondrial membrane potential (△ψm)
The△ψm, which has been suggested to be central to the apoptotic pathway (Ly et al.,2003), was measured by flow cytometry using the TMRM detection kit according to the manufacturer's instructions. PC12cells were incubated in the presence of50nM TMRM for30min at room temperature in the dark and then stored on ice until analysis by flow cytometry. For each sample,10,000particles were analyzed. The final suspension was analyzed using a FACS Calibur flow cytometer, and the results were analyzed using the CellQuest Pro software. The arithmetic mean values of the fluorescence signals in arbitrary units were determined for each sample and graphically represented. All of the experiments were repeated three times.
4. ROS detection
Dihydroethidium (DHE) was used to measure the ROS generation in cells as previously reported with slight modifications. Briefly, at the end of the treatment period, the cells were washed and incubated with2.5μM DHE (red) at37℃for30min and Hoechst33342(blue) to stain the nucleus for30min. Fluorescence microscopy was used to capture the differences in the red fluorescence intensities. The examination was performed with a laser-scanning confocal microscope (LSM510META; Carl Zeiss, Hamburg, Germany). The images were analyzed with the Image-Pro Plus software (version6.0). The mean fluorescence intensity of each cell was calculated, and the total cell emission signals per field were averaged for data analysis.
5. Stable transfection of PC12cells with EF1A-eGFP.
To distinguish the normal PC12cells from the lead-induced PC12cells in the direct cell-to-cell contact system, some of the PC12cells were transfected with the EF1A-eGFP vector (GFP-PC12) and selected with400μg/ml G418. The transfections were performed according to the standard protocol from Cyagen. The expression of GFP was identified by fluorescence microscopy and flow cytometry analysis.
6. Co-culture system to determine the bystander effects of lead-induced PC12cells
6.1Lead acetate-induced PC12-GFP cells co-cultured with PC12-GFP (Pb2+) cells
The GFP-PC12cells were exposed to different concentrations (0.1μM,1μM,10μM, and100μM) of lead acetate (dissolved in pure water) after incubation for24h. The exposures were performed in normal growth medium. The control cells were treated with5mM NAC, which is commonly used to identify and inhibit ROS, for1h and then exposed to10μM lead acetate for24h (GFP-PC12+NAC+Pb). The cells were harvested with0.025%trypsin and0.02%EDTA (dissolved in PBS and degermed).
6.2The bystander effects of PC12-GFP (Pb2+) cells is determined by direct cell to cell contact system.
The GFP-PC12cells (1×103cells/ml) were co-cultured with PC12cells (5×103cells/ml) in three groups. First, GFP-PC12cells co-cultured with PC12cells were assumed to be the control group (GFP-PC12+PC12). GFP-PC12(Pb2+) cells co-cultured with PC12cells (GFP-PC12(Pb2+)+PC12) were used to determine the bystander effects of the induced cells. To determine the effect of CBX, an inhibitor of gap junction, on the bystander effect, GFP-PC12(Pb2+) cells were co-cultured with PC12cells in medium with100μM CBX (GFP-PC12(Pb2+)+PC12+CBX), The cells were harvested after24h of co-culture.
7. Parachute assay for gap junction function
To confirm the function of gap junctions, which was hypothesized to underline the bystander effect, in a co-culture system, the parachute assay was used. To avoid overlaps between the green fluorescences of GFP and calcein-AM, which is converted intracellularly into the gap junction-permeable dye calcein. Briefly, before co-culture, GFP-PC12(Pb2+) cells were double-labeled with5μM CM-Dil, a membrane dye that does not spread to coupled cells, and5μM calcein-acetoxymethyl ester, which is converted intracellularly into the gap junction-permeable dye calcein. After incubation for30min, the GFP-PC12(Pb2+) cells were then trypsinized, diluted to500cells/ml, and used as the donor cells. Two groups of PC12cells were plated in12-well plates as receptor cells:one group of cells was incubated with1ml of the donor solution, and the other group of cells was incubated with1ml of the donor solution supplemented with100μM CBX, which inhibits the formation of gap junctions without affecting the activity of connexin. The cells were incubated for approximately4h in5%CO2at37℃and then examined with an inverted fluorescence microscope.
8. Statistic
The above test needs to be performed at least three times. The results for the between-group tests are presented by "mean±standard deviation". The between-group tests are divided into two cases according to the type of variance. When homogeneity of variance holds, we use one-way analysis of variance and LSD method forpairwise comparison. When heterogeneity of variance holds, we use Welch method and Dunnett-T3for pairwise comparison. And the results for the between-group tests are presented "mean±standard deviation". We use SPSS19.0to analyze the data. The difference is statistically significant if "P<0.05".
The main results:
1. Lead acetate is toxic to PC12cells dose-dependentty. The toxic can be inhibited by NAC partly
After the incubation of PC12cells with different concentrations of lead acetate (0.1-100μM) for24h, the MTT assay, flow cytometry, and RT-PCR were used to detect the effects of lead acetate on PC12cells.
1.1Lead acetate is toxic to the viability of PC12cell dose-dependenlly
The cell viability was determined by the MTT assay. The viability of the control cells was normalized to1, and the viabilities of the other cells are expressed as percentages compared with that of the control. The exposure to1μM of lead acetate significantly (0.01
To determine the exact concentration of lead acetate for use in the subsequent studies, we performed RT-PCR to detect the gene expression levels of Bax and Bcl-2in PC12cells after treatment with lead acetate at different concentrations. All of the RT-PCR results were standardized to the levels observed in normal PC12cells (normalized to1).The RT-PCR analysis demonstrated that the expression of Bax mRNA was significantly (P<0.01,n=3) increased to3.55,3.18, and4.42after treatment with1μM,10μM, and100μM lead acetate, respectively, compared with the untreated PC12cells; however, the0.1μM group did not exhibit a significant difference. However, the differences between these treatment groups were not significant. The expression levels of Bcl-2mRNA were decreased significantly in the10μM and100μM groups (33.3%±0.55%and4.42%±0.14%, respectively, P<0.01, n=3). The analysis of the express ion levels of Bax and Bcl-2mRNA also suggests that a lead concentration of10μM is sufficient to induce cytotoxic effects in PC12cells.
1.3Lead acetate is toxic to the apoptosis rate of PC12cells dose-dependently. NAC can inhibit the apoptosis partly.
Tthe flow cytometry analysis revealed that the percentage of apoptosis in the untreated PC12cell population was0.40%±0.46%, and significantly higher percentages of PC12cells underwent apoptosis after treatment with0.1μM,1μM,10μM, and100μM(1.33%±0.42%,5.40%±0.51%,38.33%±3.06%, and,46.67%±4.51%, respectively). The percentages of apoptotic cells in the10μM and100μM groups were not significantly different. However, a further study showed that18.67%±2.08%of the cells in the100μM group died, whereas a lower percentage of cells (9.97%±1.95%) died in the10μM group (P<0.01, n=3). Consistent with the MTT and RT-PCR results, a lead acetate concentration of10μM was selected for the subsequent studies. NAC, a commonly used inhibitor of ROS generation, was used to determine the probable mechanism underlying lead-induced apoptosis. The data showed that pre-treatment with5mM NAC decreased the percentage of apoptotic cells to14.33%±1.53%and the percentage of dead cells was to5.13%±0.81%. This result indicates that NAC partly inhibits the apoptosis induced by lead acetate.
1.4Effect of lead acetate on ROS generation
The immunofluorescent staining shown that less PC12cells (Hoechst33342, blue) express ROS (red), and the cells exhibit a low fluorescence prior to exposure to lead acetate. After treatment with10μM lead acetate for24h, the ROS generation increased markedly. It is interesting that pre-treatment with5nM NAC for30min can inhibit ROS generation in PC12cells that are exposed to lead acetate.
To distinguish the exposed cells and unexposed PC12cells, the former were successfully and stably transfected with the EF1A-EGFP vector (GFP-PC12) before induced, and approximately99%of the GFP-PC12cells were found to be GFP-positive, as evaluated using an inverted fluorescence microscope and flow cytometry.
To exclude the effect of transfected GFP on ROS generation in PC12cells, fluorescence microscopy was used to capture the fluorescence intensities associated with ROS generation in PC12cells after transfection with GFP. Compared with normal PC12cells (normalized to1), the ROS generation levels in GFP-PC12cells, GFP-PC12+Pb cells, and GFP-PC12+NAC+Pb cells were0.92±0.15,3.97±0.47and1.09±0.16, respectively. These data show that transfected GFP has no effect on ROS generation and that GFP-PC12+Pb cells produced a significantly higher amount of ROS. Pre-treatment with NAC can inhibit the subsequent ROS generation even if the cells are incubated with lead acetate at a later point in time.
1.5NAC can inhibit the toxic of lead acetate on mitochondrial membrane potential (△ψm) of PC12cells dose-dependently mainly
Fluorescent probes for monitoring△ψm are frequently used for the assessment of mitochondrial function, particularly in the context of oxidative stress and apoptosis research. The△ψm of PC12cells, GFP-PC12cells, GFP-PC12+Pb cells, and GFP-PC12+NAC+Pb cells was determined through FACS analysis using the fluorescent probe TMRM (red). The GFP-PC12+Pb cells expressed the lowest Aym, and the cells in the other groups showed the same intensity compared with the PC12group.
2. The bystander effects of GFP-PC12(Pb24) cells are mediated by GJIC and inhibited by CBX
After successful transfection, the GFP-PC12cells were incubated in normal medium containing10μM lead acetate for24h, and the GFP-PC12(Pb2+) cells were then co-cultured with normal PC12cells.
2.1CBX inhibits functional GJIC in the co-culture system
The parachute assay results demonstrate that functional gap junctions were formed between GFP-PC12(Pb2+) cells and PC12cells. The red fluorescence represents the donor cells, e.g., GFP-PC12(Pb2+) cells, and the green fluorescence demonstrated that the donor cells transmitted the signals to the receptor cells (PC12cells) through functional gap junctions. After incubation with CBX, an inhibitor of functional gap junctions, the donor cells did not convey any signals.
2.2The ROS generation in PC12cells in the co-culture system can be inhibited by CBX
Images of the immunofluorescent staining and the diagram shown indicates that minimal red fluorescence was detected in the GFP-PC12+PC12cells (normalized to1). However, almost all of the GFP-PC12(Pb2+)+PC12cells expressed a significantly higher level of red fluorescence in the nucleus (3.48±0.59). In contrast, pre-treatment with CBX resulted in a lower level of red fluorescence (1.64±0.42) compared with that expressed by the GFP-PC12(Pb2+)+PC12cells (P<0.01, n=3) and a higher level of fluorescence than that observed in the GFP-PC12+PC12cells (P<0.01,n=3).
2.3The apoptosis of PC12cells in the co-culture system can be inhibited by CBX
In the co-culture system, GFP-PC12(Pb2+) cells were GFP(+), whereas the co-cultured PC12cells were GFP(-). To detect the apoptosis of the co-cukured PC12cells, the Annexin V-PE/7-AAD apoptosis detection kit, which stains apoptosis cells a red color, was used. The Annexin V(+)GFP(-) cells represent cells that were affected by the bystander effect and thus need to be further analyzed.The Annexin V(+)GFP(-) PC12cells in the co-culture system endure the highest percentage of apoptosis (43.52%±8.07%). CBX significantly inhibits cell apoptosis (12.19%±1.13%).
2.4The changes in the mitochondrial membrane potential (△ψm) of PC12cells in the co-culture system can be inhibited by CBX
A FACS analysis was used to measure the△ψm in GFP-PC12+PC12, GFP-PC12(Pb24)+Pb, and GFP-PC12(Pb2+)+Pb+CBX cells using the fluorescent probe TMRM. In the co-culture system, only the TMRM(+)GFP(-) cells were investigated. The selected cells from the GFP-PC12(Pb2+)+Pb group exhibited the lowest△ψm, and the cells in the GFP-PC12(Pb2+)+Pb+CBX group presented a lower intensity compared with the GFP-PC12+PC12group. This finding demonstrates that CBX can inhibit the lead-induced changes in△ψm.
Conclusions:
1. Lead acetate can induce the mitochondrial pathway of apoptosis in PC12cells.
2. In co-culture system, Pb-exposed GFP-PC12cells exerted bystander effects that resulted in increased apoptosis, the generation of ROS, and the collapse of the MMP of the untreated PC12cells.
3. The bystander effects, which drive the expansion of the Pb-induced damage to neighboring cells, were determined to be dependent on functional GJIC.
引文
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