氯胺酮相关性泌尿系统损害发病机制研究
摘要
研究背景和目的
K粉主要成分为氯胺酮,近几年一些长期吸食新型毒品K粉的患者出现严重的尿频、尿急、尿痛、血尿、排尿困难、急迫性尿失禁等临床症状,暂且称之为“氯胺酮相关性泌尿系统损害(ketamine associated urinary dysfunction, KAUD)"。其发病机制尚不明确。本课题应用体外原代培养膀胱上皮细胞,研究经不同浓度的氯胺酮作用于细胞后的一系列生物学行为改变。这些生物学行为包括细胞增殖和细胞凋亡,同时在分子水平研究这些改变的分子机制,包括对凋亡相关蛋白、基因和对线粒体通路的影响等。为了进一步探究氯胺酮对于膀胱的毒性作用,我们建立大鼠KAUD动物模型,探讨氯胺酮在体内对膀胱上皮的影响,并进一步探讨其分子机制。另外,收集临床KAUD患者尿液行荧光原位杂交(fluorescence in situ hybridization, FISH)检测,从染色体水平,探讨氯胺酮对膀胱上皮细胞的影响,及KAUD与膀胱肿瘤发生的相关性。
方法
体外原代培养膀胱移行尿路上皮细胞,倒置显微镜细胞形态观察,细胞暴露于不同浓度的氯胺酮中,观察细胞形态学的改变,MTT比色法检测氯胺酮对膀胱上皮细胞增殖的影响；TUNEL法观测氯胺酮对膀胱上皮细胞凋亡率的影响。Western blot检测不同浓度氯胺酮作用下NF-κB、Bcl-2、Bax、细胞色素C、Caspase-3的蛋白表达情况,用RT-PCR技术检测经不同浓度氯胺酮作用后细胞NF-κB、Bcl-2、Bax、细胞色素C、Caspase-3基因mRNA表达水平的改变,以探讨氯胺酮对膀胱上皮细胞凋亡的作用机制。
雌性Wistar大鼠80只,体重180-220g,随机分为8组(4个实验组,4个相应的对照组),每组10只,实验组大鼠每日同一时间腹腔注射氯胺酮,注射剂量分为50mg/kg和100mg/kg,饲养时间分为1个月和3个月,分别为50mg/kg1月组,50mg/kg3月组,100mg/kg1月组,100mg/kg3月组,4个对照组分别注射等量等时间的生理盐水。观察各组大鼠刻板行为(stereotyped behavior SB)和内脏疼痛行为并进行相关评分,观察排尿次数和体重变化,建立大鼠KAUD动物模型。拉颈处死后,取大鼠膀胱组织进行HE染色观察组织结构和层次改变：采用透射电镜法检测组织细胞超微结构的影响；采用Tunel法检测各组凋亡情况。
临床中收集12例KAUD患者尿液行脱落细胞学和FISH检测,探针诊断试剂盒,包括CSP7/CSP3和GLPP16/CSP17两组双色探针。CSP7/CSP3探针分别定位于7p11.1-q11.1和3p11.1-q11.1,对应的荧光信号为红色、绿色；GLPP16/CSP17探针分别定位于9p21和17p11.1-q11.1,对应的荧光信号为红色、绿色。观察染色体改变情况,并分析异常染色体改变与吸毒量和吸毒时间之间的关系。
通过SPSS13.0软件,采用单因素方差分析,析因分析和双变量相关分析进行统计处理,P<0.05认为有统计学意义。
结果
膀胱上皮细胞原代培养并传代成功,不同浓度的氯胺酮作用膀胱上皮细胞6h、24h、48h、72h后,细胞的生长受到不同程度的抑制。不同作用时间间存在统计学差异(F=130.632,P<0.001),氯胺酮作用24h后的抑制率(平均为0.398)显著高于其他三个时间点。不同浓度之间的抑制率有显著差异(F=76.957,P<0.001),各个浓度组与阴性对照组间均存在统计学差异(P<0.001)。时间与浓度间存在交互效应(F=4.702, P<0.001)。
TUNEL法检测发现10ug/ml (18.500±1.998)、100ug/ml (31.133±4.411)、500ug/ml (39.933±6.278)、1000ug/ml(57.967±4.594)、2500ug/ml (19.533±2.203)的氯胺酮分别作用膀胱上皮细胞24h后,加药组均出现棕褐色的凋亡细胞,对照组细胞均未见或偶见阳性细胞(6.567±2.977),除10ug/ml组外(P=0.073),其他实验组和对照组均存在统计学差异(P<0.05),最高值出现在1000ug/ml组(57.967±4.594)。
WB检测不同浓度氯胺酮处理膀胱上皮细胞后,与对照组(86.330±1.528)相比,100ug/ml (64.330±5.132)、500ug/ml (55.000±5.000)、1000ug/ml (46.330±2.082)、2500ug/ml (39.000±1.000)剂量组NF-κB蛋白表达降低(P<0.05),呈剂量依赖性；100ug/ml (87.000±3.606)、500ug/ml (70.670±2.517)、1000ug/ml (56.670±3.055)、2500ug/ml (85.330±6.658)剂量组Bcl-2蛋白表达降低(P<0.05);500ug/ml (89.000±8.185)、1000ug/ml (96.670±5.033)、2500ug/ml (111.330±4.163)剂量组bax表达升高(P<0.05)；500ug/ml(124.330±6.429)、1000ug/ml(127.330±3.055)剂量组细胞色素C表达显著升高(P<0.05)；500ug/ml(90.670±6.658)、1000ug/ml(95.330±5.033)剂量组caspase-3蛋白表达显著增高(P<0.05)。
RT-PCR检测膀胱上皮细胞中各凋亡相关基因的表达,细胞经不同浓度的氯胺酮处理后,与对照组相比,各剂量组NF-κB均下调(P<0.001),并呈递减趋势；Bcl-2mRNA的表达明显受到抑制(P<0.001)；10ug/ml(2.246±0.204)、100ug/ml(1.428±0.212)和1000ug/ml(2.730±0.558)剂量组Bax与对照组无统计学差异(P>0.05),200ug/ml(3.757±0.419)、500ug/ml(3.082±0.449)、2500ug/ml(3.293±0.361)、5000ug/ml(5.246±0.258)组均存在统计学差异(P<0.05)；细胞色素C的表达出现不稳定趋势,200ug/ml(1.905±0.337)、2500ug/ml(1.981±0.309)、5000ug/ml(1.998±0.391)三个剂量组与对照组比较有统计学差异,10ug/ml(0.960±0.120)、100ug/ml(0.745±0.138)、500ug/ml(1.452±0.294)、1000ug/ml(1.228±0.086)四个剂量组与对照组相比无显著差异(P>0.05)；caspase-3mRNA的表达从有200ug/ml剂量组开始有上调趋势(P<0.001),且随着药物作用浓度增高,表达逐渐增强。
成功建立大鼠KAUD动物模型。对照组注射生理盐水后,行为活动与正常大鼠无异,正常饮水进食；实验组注射氯胺酮后开始出现行为和活动异常,主要表现为不自主的扭头、点头、啮齿(磨牙)和自发转圈运动等刻板行为。与对照组相比,实验组大鼠体重明显受抑制,实验组和对照组有显著差异(F=100.421,P<0.001),时间点有显著性差异(F=100.421,P<0.001),剂量间无显著差异(F=0.066,P=0.798),分组和时间存在交互效应(F=9.640,P=0.003),分组和剂量无交互效应(F=0.662,P=0.419),时间和剂量无交互效应(F=0.103,P=0.749),分组、时间和剂量三个因素间不存在交互效应(F=0.166,P=0.685)
通过对实验大鼠刻板行为的观察,发现实验组大鼠注射氯胺酮后出现明显的刻板行为增多。刻板行为评分明显高于对照组,实验组和对照组有显著差异(F=791.807,P<0.001),时间点无显著性差异(F=3.657,P=0.057),剂量间存在显著差异(F=4.387,P=0.037),分组和时间存在交互效应(F=4.387,P=0.037),分组和剂量也存在交互效应(F=9.031,P=0.003),时间和剂量无交互效应(F=0.008,P=0.928),分组、时间和剂量三个因素间不存在交互效应(F=0.207,P=0.649)。
通过对实验大鼠内脏疼痛行为的观察,发现实验组大鼠注射氯胺酮后出现明显的内脏疼痛行为增多。评分明显高于对照组,实验组和对照组有显著差异(F=484.921,P<0.001),1月和3月组间有显著性差异(F=36.853,P<0.001),不同注射剂量间有显著性差异(F=75.505,P<0.001),分组和时间存在交互效应(F=25.392,P<0.001),分组和剂量也存在交互效应(F=108.892,P<0.001),时间和剂量间不存在交互效应(F=2.155,P=0.143),分组、时间和剂量三个因素间存在交互效应(F=5.115,P=0.025)。
氯胺酮注射后,大鼠排尿次数明显增多,实验组和对照组有显著差异(F=570.545,P<0.001),时间点无显著性差异(F=0.599,P=0.440),剂量间有显著性差异(F=10.184,P=0.002),分组和时间存在交互效应(F=5.387,P=0.021),分组和剂量也存在交互效应(F=10.184,P=0.002),时间和剂量间不存在交互效应(F=0.150,P=0.699),分组、时间和剂量三个因素间不存在交互效应(F=0.037,P=0.847)。
HE染色观察：实验组大鼠膀胱组织上皮细胞层损伤明显,部分上皮细胞层全层脱落形成局部溃疡。膀胱壁尤其是粘膜下层和肌层出现明显水肿、血管充血、破裂出血,粘膜固有层结缔组织和粘膜小血管周围出现大量多形核中性白细胞侵润,侵润甚至出现在膀胱全层。透射电镜结果显示,细胞胞浆浓集,染色质固缩,聚集于核膜下,并可见凋亡小体形成。
大鼠膀胱组织TUNEL凋亡检测结果：阳性细胞核呈棕黄色,染色质凝聚、浓缩,呈凋亡细胞的形态学征象,部分阳性细胞核染色质仍很疏松,部分呈圆形深染的棕黄色小体,为典型的凋亡小体。在对照组中,偶见阳性细胞,可能是膀胱细胞的正常凋亡或者是假阳性的结果,而在经氯胺酮处理组,可见明显的细胞凋亡增加的现象。实验组和对照组间凋亡情况存在显著差异(F=1548.244,P<0.001),时间点有显著差异(F=153.021,P<0.001),剂量间有显著差异(F=216.566,P<0.001),分组和时间存在交互效应(F=140.891,P<0.001),分组和剂量存在交互效应(F=232.207,P<0.001),时间和剂量不存在交互效应(F=0.438,P=0.509),分组、时间和剂量不存在交互效应(F=2.067,P=0.152)。表明氯胺酮能诱导大鼠膀胱细胞凋亡,且有时间和剂量效应。
本研究中12例患者,男9例,女3例,年龄18-34岁,平均27.5岁,吸毒时间13-40月,平均27月,每日吸毒量1.5-5克,平均3.2克。细胞学均为阴性,FISH阳性5例。另有1号和12号患者分别只有一个染色体突变,所有突变中,3、7、P16、17突变率分别为4/12、5/12、1/12、2/12。每日吸毒量与FISH间存在显著相关性(r=0.595,P=0.041),总吸毒量与FISH间存在显著相关性(r=0.808,P=0.001)。
结论
体外实验中氯胺酮对膀胱上皮细胞的增殖具有明显的抑制作用,并诱导其凋亡。
体外实验中氯胺酮可调控膀胱上皮细胞凋亡相关蛋白和mRNA的表达,其机制可能是通过下调NF-κB引起线粒体凋亡通路,Bcl-2下调,Bax上调,Bax/Bcl-2比值升高,细胞色素C外流,释放入胞浆,活化的caspase-3表达增多,从而启动细胞凋亡。
成功建立大鼠KAUD动物模型,大鼠刻板运动、内脏疼痛行为、排尿次数、体重等均发生变化。
体内实验表明氯胺酮可引起大鼠膀胱上皮损伤和凋亡。
KAUD患者尿液脱落细胞存在染色体变异,且与吸毒总量相关,但染色体突变的远期结果是否与肿瘤发生相关,尚需进一步研究。
Background and Objection:
More and more chronic recreational ketamine abusers suffer from severe frequency, urgency, painful, haematuria, dysuria and urge incontinence, which we called ketamine associated urinary dysfunction (KAUD), and nothing was known about the pathogenesis. The present study was carried out to investigate the effect of ketamine on cell culture in vitro, setup the KAUD animal model and detect the chromosome with FISH, in order to approach the molecular pathogenesis of KAUD.
Methods and materials:
Rat primary urothelium cell was cultivated in vitro. MTT assay was used to measure proliferation activity of urothelium cell by ketamine. Apoptotic cell death was determined by TUNEL-staining. The protein expressions of NF-κB, Bcl-2, Bax. Cytochrome C and Caspase-3were assessed by Western Blot. The expression of mRNA determined by reverse transcript-polymerase chain reaction (RT-PCR) assay.
80wistar rats were divided into8groups at random:50mg/kg1month,50mg/kg3months,100mg/kg1month,100mg/kg3months, and four corresponding control groups. They were exposed to ketamine by intraperitioneal injection or equivalence saline. Experiments and observations described below were performed after exposure. Stereotyped behavior, internal organs pain behavior, urination times and body weight changes were observed. Apoptosis of urothelium cell in rat bladder were measured with method of TUNEL. Morphology of bladder was observed with light microscope and electron microscope.
Urine samples of12KAUD patients were analyzed by means of cytology and fluorescence in situ hybridization. Fluorescence in situ hybridization were used to detect the abnormalities of chromosome3,7,17and p16. We analyzed the rates of abnormalities in chromosome3,7,17, p16. The probe mix used consisted of centromeric enumeration probes of chromosomes3(CEP3),7(CEP7), and17(CEP17), and locus-specific identifier probes to the9p21locus location of the p16tumor suppressor gene (LSI9p21or p16). Two DNA probes were mixed together as a set double-target FISH and paired as follows:chromosome3(rhodamine) and chromosome7(FITC), chromosome17(FITC) and p16(rhodamine). We analyses the relationship between chromosomal change and drug abuse.
Data were analysis by One-Way ANOVA, Factorial analysis and Bivariate in SPSS13.0software.
Results:
Rat primary urothelium cell modle treated with ketamine was setup successfully. Ketamine could inhibit the proliferation of urothelium cells significantly in a dose-and time-dependent manner. There was statistics difference in different action time groups (F=130.632, P<0.001) and different ketamine density groups (F=76.957, P <0.001). Interaction could be found between time and density (F=4.702, P<0.001).
TUNEL assay also showed ketamine could induce apoptosis in urothelium cells in lOug/ml (18.500±1.998),100ug/ml (31.133±4.411),500ug/ml (39.933±6.278),1000ug/ml (57.967±4.594),2500ug/ml (19.533±2.203). Apoptosis cells showed in all ketamine groups, but did not in control group (6.567±2.977). Compared with control group, there was statistics difference in all experimental group (P<0.05), except10ug/ml group (P=0.073), with maximum level in1000ug/ml group (57.967±4.594)
Ketamine could regulate the expression of apoptosis-related protein. Western blot analysis showed that the protein expression of NF-κB was down-regulated in100ug/ml (64.330±5.132),500ug/ml (55.000±5.000),1000ug/ml (46.330±2.082),2500ug/ml (39.000±1.000) groups (P<0.05). Bcl-2was down-regulated in100ug/ml (87.000±3.606),500ug/ml (70.670±2.517),1000ug/ml (56.670±3.055),2500ug/ml (85.330±6.658) groups (P<0.05), but the protein expression of Bax was up-regulated in500ug/ml (89.000±8.185),1000ug/ml (96.670±5.033),2500ug/ml (111.330±4.163) groups (P<0.05). Cytochrome C was up-regulated in500ug/ml (124.330±6.429),1000ug/ml(127.330±3.055) groups (P<0.05). Caspase-3were advanced in500ug/ml (90.670±6.658), lOOOug/ml (95.330±5.033) groups (P<0.05)
RT-PCRanalysis showed that the expression of NF-κB and Bcl-2were down-regulated in urothelium cells treated with ketamine in a dose-dependent manner (P<0.001), whereas the expression of Bax was up-regulated in200ug/ml (3.757±0.419),500ug/ml (3.082±0.449),2500ug/ml (3.293±0.361),5000ug/ml (5.246±0.258) groups(P<0.05). Cytochrome C was up-regulated in200ug/ml (1.905±0.337),2500ug/ml (1.981±0.309),5000ug/ml (1.998±0.391) groups(P<0.05). Caspase-3was up-regulated from200ug/ml to5000ug/ml groups (P<0.001).
KAUD rat modle was setup successfully. Rats body weight were inhibited obviously in ketamine groups compared with control group (F=100.421, P<0.001). Significant difference showed in different time (F=100.421, P<0.001), but without in different dose (F=0.066, P=0.798). Interaction effects were present in group and time (F=9.640, P=0.003), time and dose (F=0.103. P=0.749), but not in group and dose(F=0.662, P=0.419), and not among the three factors(F=0.166, P=0.685).
Abnormal behavior showed up after treated with ketamine. Results show significant difference in stereotyped behavior compared with control group (F=791.807, P<0.001). Significant difference showed in different dose (F=4.387, P=0.037), but without in different time (F=3.657, P=0.057). Interaction effects were present in group and time (F=4.387, P=0.037), group and dose (F=9.031, P=0.003), but not in time and dose (F=0.008, P=0.928), and not among the three factors (F=0.207, P=0.649)
Internal organs pain behavior score was higher than control group (F=484.921, P<0.001). Significant difference showed in different time (F=36.853. P<0.001) and in different dose(F=75.505, P<0.001). Interaction effects were present in group and time(F=25.392, P<0.001), group and dose(F=108.892, P<0.001), and among the three factors (F=5.115, P=0.025), but not in time and dose (F=2.155, P=0.143)
After injected with ketamine, rats urination times increased significantly (F=570.545, P<0.001). Significant difference also showed in different dose(F=10.184, P=0.002), but without in different time (F=0.599, P=0.440). Interaction effects were present in group and time (F=5.387, P=0.021), group and dose (F=10.184, P=0.002), but not in time and dose(F=0.150, P=0.699), and not among the three factors(F=0.037, P=0.847).
The observation under light microcope showed:arrangement of the urothelium cell was orderly in control group, but with the increasing of the doses and time of ketamine exposed, the urothelium cell displayed disorderly, the connections among the urothelium cell were loosened. Urothelium cell layer was danaged, lossed or desquanmated. Edema, vasocongestion and hemorrhage showed in submucosa and muscular layer. Polymorphonuclear neutrophil leukocyte can also be found in connective tissue and vessels surrounding. Picture under electron microscope displayed:the karyon shrinlced, the karyotin assembled in the edge, karyotheca was broken, mitochondria swelled, broken, apoptotic body can be found.
TUNEL assay showed ketamine could induce apoptosis in bladder urothelium cells with morphology signs(buffy positive cells, chromatin agglutination, apoptotic body) Results show significant difference between ketamine groups and control group (F=1548.244, P<0.001). Significant difference showed in different time (F=153.021, P<0.001), and in different dose(F=216.566, P<0.001). Interaction effects were present in group and time(F=140.891, P<0.001), group and dose(F=232.207, P<0.001), but not in time and dose (F=0.438. P=0.509), and not among the three factors (F=2.067, P=0.152)
5positive FISH and all negative cytology were detected in12KAUD patients, with9male and3female. The aberration rates of3,7,17, p16were4/12,5/12,1/12,2/12. There were correlation between daily dose and FISH(r=0.595, P=0.041), total dose and FISH(r=0.808, P=0.001).
Conclusion:
Ketamine could inhibit the proliferation of urothelium cells significantly and induce apoptosis in a dose-and time-dependent manner in vitro.
Ketamine could regulate the expression of apoptosis-related protein and mRNA. The mechanism may trigger mitochondria apoptosis path through the downwards regulation of NF-κB translocation reduction, which may reduce the ratio between anti-and pro-apoptotic proteins by increasing the expression of pro-apoptotic protein Bax and inducing the degradation of anti-apoptotic protein Bcl-2, and consequent the decrease of membrane potential, release of cytochrome c and activation of caspase-3 result in apoptosis.
KAUD rat modle was setup successfully. Stereotyped behavior, internal organs pain behavior, urination times and body weight changes were observed.
Ketamine could caused damage and apoptosis in rat bladder tissue in a dose-and time-dependent manner.
Chromosomal variation was found in cell of KAUD patient urine by FISH. Further study was needed to make clear whether it is correlated to tumorigenesis.
K粉主要成分为氯胺酮,近几年一些长期吸食新型毒品K粉的患者出现严重的尿频、尿急、尿痛、血尿、排尿困难、急迫性尿失禁等临床症状,暂且称之为“氯胺酮相关性泌尿系统损害(ketamine associated urinary dysfunction, KAUD)"。其发病机制尚不明确。本课题应用体外原代培养膀胱上皮细胞,研究经不同浓度的氯胺酮作用于细胞后的一系列生物学行为改变。这些生物学行为包括细胞增殖和细胞凋亡,同时在分子水平研究这些改变的分子机制,包括对凋亡相关蛋白、基因和对线粒体通路的影响等。为了进一步探究氯胺酮对于膀胱的毒性作用,我们建立大鼠KAUD动物模型,探讨氯胺酮在体内对膀胱上皮的影响,并进一步探讨其分子机制。另外,收集临床KAUD患者尿液行荧光原位杂交(fluorescence in situ hybridization, FISH)检测,从染色体水平,探讨氯胺酮对膀胱上皮细胞的影响,及KAUD与膀胱肿瘤发生的相关性。
方法
体外原代培养膀胱移行尿路上皮细胞,倒置显微镜细胞形态观察,细胞暴露于不同浓度的氯胺酮中,观察细胞形态学的改变,MTT比色法检测氯胺酮对膀胱上皮细胞增殖的影响；TUNEL法观测氯胺酮对膀胱上皮细胞凋亡率的影响。Western blot检测不同浓度氯胺酮作用下NF-κB、Bcl-2、Bax、细胞色素C、Caspase-3的蛋白表达情况,用RT-PCR技术检测经不同浓度氯胺酮作用后细胞NF-κB、Bcl-2、Bax、细胞色素C、Caspase-3基因mRNA表达水平的改变,以探讨氯胺酮对膀胱上皮细胞凋亡的作用机制。
雌性Wistar大鼠80只,体重180-220g,随机分为8组(4个实验组,4个相应的对照组),每组10只,实验组大鼠每日同一时间腹腔注射氯胺酮,注射剂量分为50mg/kg和100mg/kg,饲养时间分为1个月和3个月,分别为50mg/kg1月组,50mg/kg3月组,100mg/kg1月组,100mg/kg3月组,4个对照组分别注射等量等时间的生理盐水。观察各组大鼠刻板行为(stereotyped behavior SB)和内脏疼痛行为并进行相关评分,观察排尿次数和体重变化,建立大鼠KAUD动物模型。拉颈处死后,取大鼠膀胱组织进行HE染色观察组织结构和层次改变：采用透射电镜法检测组织细胞超微结构的影响；采用Tunel法检测各组凋亡情况。
临床中收集12例KAUD患者尿液行脱落细胞学和FISH检测,探针诊断试剂盒,包括CSP7/CSP3和GLPP16/CSP17两组双色探针。CSP7/CSP3探针分别定位于7p11.1-q11.1和3p11.1-q11.1,对应的荧光信号为红色、绿色；GLPP16/CSP17探针分别定位于9p21和17p11.1-q11.1,对应的荧光信号为红色、绿色。观察染色体改变情况,并分析异常染色体改变与吸毒量和吸毒时间之间的关系。
通过SPSS13.0软件,采用单因素方差分析,析因分析和双变量相关分析进行统计处理,P<0.05认为有统计学意义。
结果
膀胱上皮细胞原代培养并传代成功,不同浓度的氯胺酮作用膀胱上皮细胞6h、24h、48h、72h后,细胞的生长受到不同程度的抑制。不同作用时间间存在统计学差异(F=130.632,P<0.001),氯胺酮作用24h后的抑制率(平均为0.398)显著高于其他三个时间点。不同浓度之间的抑制率有显著差异(F=76.957,P<0.001),各个浓度组与阴性对照组间均存在统计学差异(P<0.001)。时间与浓度间存在交互效应(F=4.702, P<0.001)。
TUNEL法检测发现10ug/ml (18.500±1.998)、100ug/ml (31.133±4.411)、500ug/ml (39.933±6.278)、1000ug/ml(57.967±4.594)、2500ug/ml (19.533±2.203)的氯胺酮分别作用膀胱上皮细胞24h后,加药组均出现棕褐色的凋亡细胞,对照组细胞均未见或偶见阳性细胞(6.567±2.977),除10ug/ml组外(P=0.073),其他实验组和对照组均存在统计学差异(P<0.05),最高值出现在1000ug/ml组(57.967±4.594)。
WB检测不同浓度氯胺酮处理膀胱上皮细胞后,与对照组(86.330±1.528)相比,100ug/ml (64.330±5.132)、500ug/ml (55.000±5.000)、1000ug/ml (46.330±2.082)、2500ug/ml (39.000±1.000)剂量组NF-κB蛋白表达降低(P<0.05),呈剂量依赖性；100ug/ml (87.000±3.606)、500ug/ml (70.670±2.517)、1000ug/ml (56.670±3.055)、2500ug/ml (85.330±6.658)剂量组Bcl-2蛋白表达降低(P<0.05);500ug/ml (89.000±8.185)、1000ug/ml (96.670±5.033)、2500ug/ml (111.330±4.163)剂量组bax表达升高(P<0.05)；500ug/ml(124.330±6.429)、1000ug/ml(127.330±3.055)剂量组细胞色素C表达显著升高(P<0.05)；500ug/ml(90.670±6.658)、1000ug/ml(95.330±5.033)剂量组caspase-3蛋白表达显著增高(P<0.05)。
RT-PCR检测膀胱上皮细胞中各凋亡相关基因的表达,细胞经不同浓度的氯胺酮处理后,与对照组相比,各剂量组NF-κB均下调(P<0.001),并呈递减趋势；Bcl-2mRNA的表达明显受到抑制(P<0.001)；10ug/ml(2.246±0.204)、100ug/ml(1.428±0.212)和1000ug/ml(2.730±0.558)剂量组Bax与对照组无统计学差异(P>0.05),200ug/ml(3.757±0.419)、500ug/ml(3.082±0.449)、2500ug/ml(3.293±0.361)、5000ug/ml(5.246±0.258)组均存在统计学差异(P<0.05)；细胞色素C的表达出现不稳定趋势,200ug/ml(1.905±0.337)、2500ug/ml(1.981±0.309)、5000ug/ml(1.998±0.391)三个剂量组与对照组比较有统计学差异,10ug/ml(0.960±0.120)、100ug/ml(0.745±0.138)、500ug/ml(1.452±0.294)、1000ug/ml(1.228±0.086)四个剂量组与对照组相比无显著差异(P>0.05)；caspase-3mRNA的表达从有200ug/ml剂量组开始有上调趋势(P<0.001),且随着药物作用浓度增高,表达逐渐增强。
成功建立大鼠KAUD动物模型。对照组注射生理盐水后,行为活动与正常大鼠无异,正常饮水进食；实验组注射氯胺酮后开始出现行为和活动异常,主要表现为不自主的扭头、点头、啮齿(磨牙)和自发转圈运动等刻板行为。与对照组相比,实验组大鼠体重明显受抑制,实验组和对照组有显著差异(F=100.421,P<0.001),时间点有显著性差异(F=100.421,P<0.001),剂量间无显著差异(F=0.066,P=0.798),分组和时间存在交互效应(F=9.640,P=0.003),分组和剂量无交互效应(F=0.662,P=0.419),时间和剂量无交互效应(F=0.103,P=0.749),分组、时间和剂量三个因素间不存在交互效应(F=0.166,P=0.685)
通过对实验大鼠刻板行为的观察,发现实验组大鼠注射氯胺酮后出现明显的刻板行为增多。刻板行为评分明显高于对照组,实验组和对照组有显著差异(F=791.807,P<0.001),时间点无显著性差异(F=3.657,P=0.057),剂量间存在显著差异(F=4.387,P=0.037),分组和时间存在交互效应(F=4.387,P=0.037),分组和剂量也存在交互效应(F=9.031,P=0.003),时间和剂量无交互效应(F=0.008,P=0.928),分组、时间和剂量三个因素间不存在交互效应(F=0.207,P=0.649)。
通过对实验大鼠内脏疼痛行为的观察,发现实验组大鼠注射氯胺酮后出现明显的内脏疼痛行为增多。评分明显高于对照组,实验组和对照组有显著差异(F=484.921,P<0.001),1月和3月组间有显著性差异(F=36.853,P<0.001),不同注射剂量间有显著性差异(F=75.505,P<0.001),分组和时间存在交互效应(F=25.392,P<0.001),分组和剂量也存在交互效应(F=108.892,P<0.001),时间和剂量间不存在交互效应(F=2.155,P=0.143),分组、时间和剂量三个因素间存在交互效应(F=5.115,P=0.025)。
氯胺酮注射后,大鼠排尿次数明显增多,实验组和对照组有显著差异(F=570.545,P<0.001),时间点无显著性差异(F=0.599,P=0.440),剂量间有显著性差异(F=10.184,P=0.002),分组和时间存在交互效应(F=5.387,P=0.021),分组和剂量也存在交互效应(F=10.184,P=0.002),时间和剂量间不存在交互效应(F=0.150,P=0.699),分组、时间和剂量三个因素间不存在交互效应(F=0.037,P=0.847)。
HE染色观察：实验组大鼠膀胱组织上皮细胞层损伤明显,部分上皮细胞层全层脱落形成局部溃疡。膀胱壁尤其是粘膜下层和肌层出现明显水肿、血管充血、破裂出血,粘膜固有层结缔组织和粘膜小血管周围出现大量多形核中性白细胞侵润,侵润甚至出现在膀胱全层。透射电镜结果显示,细胞胞浆浓集,染色质固缩,聚集于核膜下,并可见凋亡小体形成。
大鼠膀胱组织TUNEL凋亡检测结果：阳性细胞核呈棕黄色,染色质凝聚、浓缩,呈凋亡细胞的形态学征象,部分阳性细胞核染色质仍很疏松,部分呈圆形深染的棕黄色小体,为典型的凋亡小体。在对照组中,偶见阳性细胞,可能是膀胱细胞的正常凋亡或者是假阳性的结果,而在经氯胺酮处理组,可见明显的细胞凋亡增加的现象。实验组和对照组间凋亡情况存在显著差异(F=1548.244,P<0.001),时间点有显著差异(F=153.021,P<0.001),剂量间有显著差异(F=216.566,P<0.001),分组和时间存在交互效应(F=140.891,P<0.001),分组和剂量存在交互效应(F=232.207,P<0.001),时间和剂量不存在交互效应(F=0.438,P=0.509),分组、时间和剂量不存在交互效应(F=2.067,P=0.152)。表明氯胺酮能诱导大鼠膀胱细胞凋亡,且有时间和剂量效应。
本研究中12例患者,男9例,女3例,年龄18-34岁,平均27.5岁,吸毒时间13-40月,平均27月,每日吸毒量1.5-5克,平均3.2克。细胞学均为阴性,FISH阳性5例。另有1号和12号患者分别只有一个染色体突变,所有突变中,3、7、P16、17突变率分别为4/12、5/12、1/12、2/12。每日吸毒量与FISH间存在显著相关性(r=0.595,P=0.041),总吸毒量与FISH间存在显著相关性(r=0.808,P=0.001)。
结论
体外实验中氯胺酮对膀胱上皮细胞的增殖具有明显的抑制作用,并诱导其凋亡。
体外实验中氯胺酮可调控膀胱上皮细胞凋亡相关蛋白和mRNA的表达,其机制可能是通过下调NF-κB引起线粒体凋亡通路,Bcl-2下调,Bax上调,Bax/Bcl-2比值升高,细胞色素C外流,释放入胞浆,活化的caspase-3表达增多,从而启动细胞凋亡。
成功建立大鼠KAUD动物模型,大鼠刻板运动、内脏疼痛行为、排尿次数、体重等均发生变化。
体内实验表明氯胺酮可引起大鼠膀胱上皮损伤和凋亡。
KAUD患者尿液脱落细胞存在染色体变异,且与吸毒总量相关,但染色体突变的远期结果是否与肿瘤发生相关,尚需进一步研究。
Background and Objection:
More and more chronic recreational ketamine abusers suffer from severe frequency, urgency, painful, haematuria, dysuria and urge incontinence, which we called ketamine associated urinary dysfunction (KAUD), and nothing was known about the pathogenesis. The present study was carried out to investigate the effect of ketamine on cell culture in vitro, setup the KAUD animal model and detect the chromosome with FISH, in order to approach the molecular pathogenesis of KAUD.
Methods and materials:
Rat primary urothelium cell was cultivated in vitro. MTT assay was used to measure proliferation activity of urothelium cell by ketamine. Apoptotic cell death was determined by TUNEL-staining. The protein expressions of NF-κB, Bcl-2, Bax. Cytochrome C and Caspase-3were assessed by Western Blot. The expression of mRNA determined by reverse transcript-polymerase chain reaction (RT-PCR) assay.
80wistar rats were divided into8groups at random:50mg/kg1month,50mg/kg3months,100mg/kg1month,100mg/kg3months, and four corresponding control groups. They were exposed to ketamine by intraperitioneal injection or equivalence saline. Experiments and observations described below were performed after exposure. Stereotyped behavior, internal organs pain behavior, urination times and body weight changes were observed. Apoptosis of urothelium cell in rat bladder were measured with method of TUNEL. Morphology of bladder was observed with light microscope and electron microscope.
Urine samples of12KAUD patients were analyzed by means of cytology and fluorescence in situ hybridization. Fluorescence in situ hybridization were used to detect the abnormalities of chromosome3,7,17and p16. We analyzed the rates of abnormalities in chromosome3,7,17, p16. The probe mix used consisted of centromeric enumeration probes of chromosomes3(CEP3),7(CEP7), and17(CEP17), and locus-specific identifier probes to the9p21locus location of the p16tumor suppressor gene (LSI9p21or p16). Two DNA probes were mixed together as a set double-target FISH and paired as follows:chromosome3(rhodamine) and chromosome7(FITC), chromosome17(FITC) and p16(rhodamine). We analyses the relationship between chromosomal change and drug abuse.
Data were analysis by One-Way ANOVA, Factorial analysis and Bivariate in SPSS13.0software.
Results:
Rat primary urothelium cell modle treated with ketamine was setup successfully. Ketamine could inhibit the proliferation of urothelium cells significantly in a dose-and time-dependent manner. There was statistics difference in different action time groups (F=130.632, P<0.001) and different ketamine density groups (F=76.957, P <0.001). Interaction could be found between time and density (F=4.702, P<0.001).
TUNEL assay also showed ketamine could induce apoptosis in urothelium cells in lOug/ml (18.500±1.998),100ug/ml (31.133±4.411),500ug/ml (39.933±6.278),1000ug/ml (57.967±4.594),2500ug/ml (19.533±2.203). Apoptosis cells showed in all ketamine groups, but did not in control group (6.567±2.977). Compared with control group, there was statistics difference in all experimental group (P<0.05), except10ug/ml group (P=0.073), with maximum level in1000ug/ml group (57.967±4.594)
Ketamine could regulate the expression of apoptosis-related protein. Western blot analysis showed that the protein expression of NF-κB was down-regulated in100ug/ml (64.330±5.132),500ug/ml (55.000±5.000),1000ug/ml (46.330±2.082),2500ug/ml (39.000±1.000) groups (P<0.05). Bcl-2was down-regulated in100ug/ml (87.000±3.606),500ug/ml (70.670±2.517),1000ug/ml (56.670±3.055),2500ug/ml (85.330±6.658) groups (P<0.05), but the protein expression of Bax was up-regulated in500ug/ml (89.000±8.185),1000ug/ml (96.670±5.033),2500ug/ml (111.330±4.163) groups (P<0.05). Cytochrome C was up-regulated in500ug/ml (124.330±6.429),1000ug/ml(127.330±3.055) groups (P<0.05). Caspase-3were advanced in500ug/ml (90.670±6.658), lOOOug/ml (95.330±5.033) groups (P<0.05)
RT-PCRanalysis showed that the expression of NF-κB and Bcl-2were down-regulated in urothelium cells treated with ketamine in a dose-dependent manner (P<0.001), whereas the expression of Bax was up-regulated in200ug/ml (3.757±0.419),500ug/ml (3.082±0.449),2500ug/ml (3.293±0.361),5000ug/ml (5.246±0.258) groups(P<0.05). Cytochrome C was up-regulated in200ug/ml (1.905±0.337),2500ug/ml (1.981±0.309),5000ug/ml (1.998±0.391) groups(P<0.05). Caspase-3was up-regulated from200ug/ml to5000ug/ml groups (P<0.001).
KAUD rat modle was setup successfully. Rats body weight were inhibited obviously in ketamine groups compared with control group (F=100.421, P<0.001). Significant difference showed in different time (F=100.421, P<0.001), but without in different dose (F=0.066, P=0.798). Interaction effects were present in group and time (F=9.640, P=0.003), time and dose (F=0.103. P=0.749), but not in group and dose(F=0.662, P=0.419), and not among the three factors(F=0.166, P=0.685).
Abnormal behavior showed up after treated with ketamine. Results show significant difference in stereotyped behavior compared with control group (F=791.807, P<0.001). Significant difference showed in different dose (F=4.387, P=0.037), but without in different time (F=3.657, P=0.057). Interaction effects were present in group and time (F=4.387, P=0.037), group and dose (F=9.031, P=0.003), but not in time and dose (F=0.008, P=0.928), and not among the three factors (F=0.207, P=0.649)
Internal organs pain behavior score was higher than control group (F=484.921, P<0.001). Significant difference showed in different time (F=36.853. P<0.001) and in different dose(F=75.505, P<0.001). Interaction effects were present in group and time(F=25.392, P<0.001), group and dose(F=108.892, P<0.001), and among the three factors (F=5.115, P=0.025), but not in time and dose (F=2.155, P=0.143)
After injected with ketamine, rats urination times increased significantly (F=570.545, P<0.001). Significant difference also showed in different dose(F=10.184, P=0.002), but without in different time (F=0.599, P=0.440). Interaction effects were present in group and time (F=5.387, P=0.021), group and dose (F=10.184, P=0.002), but not in time and dose(F=0.150, P=0.699), and not among the three factors(F=0.037, P=0.847).
The observation under light microcope showed:arrangement of the urothelium cell was orderly in control group, but with the increasing of the doses and time of ketamine exposed, the urothelium cell displayed disorderly, the connections among the urothelium cell were loosened. Urothelium cell layer was danaged, lossed or desquanmated. Edema, vasocongestion and hemorrhage showed in submucosa and muscular layer. Polymorphonuclear neutrophil leukocyte can also be found in connective tissue and vessels surrounding. Picture under electron microscope displayed:the karyon shrinlced, the karyotin assembled in the edge, karyotheca was broken, mitochondria swelled, broken, apoptotic body can be found.
TUNEL assay showed ketamine could induce apoptosis in bladder urothelium cells with morphology signs(buffy positive cells, chromatin agglutination, apoptotic body) Results show significant difference between ketamine groups and control group (F=1548.244, P<0.001). Significant difference showed in different time (F=153.021, P<0.001), and in different dose(F=216.566, P<0.001). Interaction effects were present in group and time(F=140.891, P<0.001), group and dose(F=232.207, P<0.001), but not in time and dose (F=0.438. P=0.509), and not among the three factors (F=2.067, P=0.152)
5positive FISH and all negative cytology were detected in12KAUD patients, with9male and3female. The aberration rates of3,7,17, p16were4/12,5/12,1/12,2/12. There were correlation between daily dose and FISH(r=0.595, P=0.041), total dose and FISH(r=0.808, P=0.001).
Conclusion:
Ketamine could inhibit the proliferation of urothelium cells significantly and induce apoptosis in a dose-and time-dependent manner in vitro.
Ketamine could regulate the expression of apoptosis-related protein and mRNA. The mechanism may trigger mitochondria apoptosis path through the downwards regulation of NF-κB translocation reduction, which may reduce the ratio between anti-and pro-apoptotic proteins by increasing the expression of pro-apoptotic protein Bax and inducing the degradation of anti-apoptotic protein Bcl-2, and consequent the decrease of membrane potential, release of cytochrome c and activation of caspase-3 result in apoptosis.
KAUD rat modle was setup successfully. Stereotyped behavior, internal organs pain behavior, urination times and body weight changes were observed.
Ketamine could caused damage and apoptosis in rat bladder tissue in a dose-and time-dependent manner.
Chromosomal variation was found in cell of KAUD patient urine by FISH. Further study was needed to make clear whether it is correlated to tumorigenesis.
引文
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