氯胺酮性膀胱纤维化中TGF-β1调控膀胱上皮细胞EMT的实验研究

发布时间：2018-08-22 19:19

【摘要】：背景长期滥用毒品氯胺酮(ketamine)会引发膀胱间质炎症(ketamine-induced cystitis,KC),主要临床表现为尿频、尿急、尿痛、排尿困难及急迫性尿失禁等。膀胱纤维化是中晚期KC患者的严重并发症。膀胱壁由于纤维结缔组织积聚而发生重塑,引起膀胱纤维化、挛缩,使其变为小容量、高压力的低顺应性膀胱,导致膀胱贮尿和排尿功能障碍,进一步发展会导致上尿路积水和肾功能损害,危及生命。目前少有研究涉及ketamine引发膀胱纤维化的机制。上皮-间质转化(epithelial-mesenchymal transitions,EMT)是指上皮细胞失去极性、在形态学上向间质细胞转变的生物学过程。目前已证实EMT广泛参与肾脏、肝脏、肠壁及肺脏的纤维化,并已成为有效终止、逆转脏器纤维化的重要靶点,然而其在膀胱纤维化中的作用尚未见报道。本项目拟从EMT介导器官纤维化入手,结合体内外模型,通过分子机制研究阐明TGF-β1信号通路介导的EMT在KC纤维化发生发展中的机制。方法1.建立KC大鼠模型并探讨EMT与膀胱纤维化的关系雌性Sprague-Dawley(SD)大鼠18只,体重180-200g,随机分为3组,每组6只,分别为对照组(Control),ketamine低剂量组(LK,30mg/kg),ketamine高剂量组(HK,60mg/kg)。每日腹腔注射ketamine溶液或生理盐水,持续16周。采用代谢笼记录24h饮水量和尿量。采用排尿印迹法评估尿频情况。对大鼠行尿动力检查,记录排尿收缩次数、逼尿肌不稳定收缩次数、膀胱内压力(基线值,阈值,最大值)、排尿量、膀胱最大容量,计算膀胱顺应性。处死大鼠后取膀胱组织,制作石蜡切片,HE及Masson染色评估膀胱上皮损害及间质纤维化情况,免疫组化实验评估 E-cadherin、Collageon I、Vimentin、Fibronectin 表达情况,E-cadherin-FSPl免疫荧光共定位评估膀胱上皮EMT发生情况。2.在KC大鼠模型中,明确TGF-β1特异性阻断剂SB505124对EMT及膀胱纤维化的调节作用雌性SD大鼠24只,体重180-200g,随机分为4组,每组6只,分别为对照组(control),ketamine(KET,60mg/kg)组,ketamine+SB505124(SB)组,戒断组(abstinence组)。SB组在注射ketamine的基础上每周腹腔注射SB505124(10 mg/kg)一次,abstinence组注射ketamine后12周戒断4周。排尿印记、尿动力检查、HE染色、Masson染色、免疫组化分析同第一部分,免疫荧光双标检测 E-cadherin-FSP1、E-cadherin-α-SMA 共表达情况。3.Ketamine通过TGF-β1调节人膀胱上皮细胞EMT的分子机制研究培养人永生化膀胱上皮(SV-HUC-1)细胞,CCK-8法检测ketamine(5-125μg/ml)对细胞活力的影响。Ketamine(125 μg/ml)刺激 SV-HUC-1 细胞 72h,相差显微镜观察细胞形态变化。Ketamine(125 μg/ml)刺激细胞24h,荧光定量 PCR 法检测 EMT 标志物(E-cadherin、FSP1、Vimentin、Fibronectin、α-SMA)表达。Ketamine(5 to 125μg/mL)刺激细胞 48h,Western Blot 检测 E-cadherin、Fibronectin、α-SMA 蛋白表达水平。Ketamine(5 to 125μg/mL)刺激细胞不同时间,ELISA检测上清液TGF-β1水平变化。SB505124阻断TGF-β1信号通路,Western Blot检测ketamine对EMT的诱导作用。荧光定量PCR法检测TGF-β1 下游信号分子(Tβ-R1,Tβ-R2,Snail,Slug,Twist)。结果1.大鼠经过16周ketamine暴露后,24小时饮水量和尿量在control组、LK组、HK组间无明显差异。LK组和HK组6小时排尿次数较对照组显著增多,HK组更为明显。尿动力学结果显示,长期滥用ketamine后,大鼠的排尿频率、逼尿肌无抑制收缩次数、膀胱最大压力均明显升高,而膀胱最大容量和膀胱顺应性明显降低,且随接ketamine剂量的增加而更加严重。HE染色结果显示:与control组相比,LK组、HK组上皮层明显变薄甚至全层缺失形成溃疡,黏膜下见不同程度的血管扩张。Masson染色结果显示:与control组相比,LK组、HK组膀胱肌层中存在大量胶原纤维,HK组更加严重。免疫组化结果证实实验组 E-cadherin 水平明显降低,Collagen I、Vimentin 和 Fibronectin 蛋白明显升高,且HK组较LK组变化更加明显。免疫荧光双标结果显示,实验组膀胱上皮层出现E-cadherin+FSP1+细胞,提示EMT的存在。免疫组化示TGF-β1在LK和HK组膀胱上皮表达显著升高。2.大鼠经过16周药物干预后,24小时饮水量和尿量在control组、KET组、SB组、abstinence组无明显差异。与KET组相比,SB组排尿次数显著降低,而abstinence组无明显变化。SB505124干预后,大鼠的排尿频率、逼尿肌无抑制收缩次数、膀胱最大压力较KET组均明显降低,膀胱最大容量和膀胱顺应性也得到不同程度的改善,而abstinence组这些指标与KET组无明显差异。HE染色和Masson染色结果显示:与KET组相比,SB组膀胱上皮及间质纤维化均得到改善。免疫组化结果表明,SB505124可以显著抑制ketamine对E-cadherin、Collagen I、Vimentin和Fibronectin蛋白表达的影响。免疫荧光双标实验显示:与 KET 组相比,SB 组 E-cadherin+FSP1+细胞和 E-cadherin+α-SMA+细胞均明显减少,提示SB505124可以抑制ketamine诱导EMT。戒断4周不能明显逆转ketamine造成的膀胱损害。3.Ketamine(5-125μg/ml)刺激 SV-HUC-1 细胞 24h 和 48h,细胞活力无明显降低。Ketamine刺激SV-HUC-1细胞72 h,细胞形态由典型的鹅卵石样转变为纺锤样。qPCR显示ketamine显著下调E-cadherin表达,上调间质标志物(FSP1、Vimentin、Fibronectin、α-SMA)的表达。Western Blot 验证 ketamine降低E-cadherin蛋白水平,上调Fibronectin、α-SMA蛋白水平,具有浓度依赖效应。ELISA实验表明,ketamine可以上调TGF-β1表达,具有时间和浓度依赖效应。SB505124明显降低ketamine诱导EMT的能力。qPCR显示ketamine增强 TGF-β1 下游信号分子(Tβ-R1,Tβ-R2,Snail,Slug,Twist)的表达。结论1.长期滥用氯胺酮可以导致大鼠膀胱容量降低、逼尿肌不稳定收缩、膀胱顺应性降低等膀胱功能障碍,病理上表现为膀胱上皮破坏和间质纤维化。2.氯胺酮性膀胱纤维化中存在EMT现象,TGF-β1特异性阻断剂SB505124可以抑制EMT和间质纤维化,EMT可能参与膀胱纤维化。3.氯胺酮在5-125 μg/ml剂量范围对人膀胱上皮细胞的活力无明显影响。氯胺酮可以通过TGF-β1信号通路诱导人膀胱上皮细胞发生EMT。4.氯胺酮刺激人膀胱上皮细胞后,可以上调TGF-β1下游信号分子(Snail,Slug,Twist)表达。5.TβR-1可能是KC纤维化的治疗靶点。
[Abstract]:BACKGROUND Long-term abuse of ketamine can induce interstitial inflammation of the bladder (KC). The main clinical manifestations are frequent urination, urgency, pain, dysuria and urgent incontinence. Cystic fibrosis, contracture, small volume, high pressure, low compliance of the bladder, leading to bladder storage and urination dysfunction, further development will lead to upper urinary tract hydrops and renal function damage, life-threatening. Ns, EMT) refers to the biological process of epithelial cells losing polarity and transforming into interstitial cells morphologically. It has been proved that EMT is widely involved in the fibrosis of kidney, liver, intestinal wall and lung, and has become an important target for effectively terminating and reversing organ fibrosis. However, its role in bladder fibrosis has not been reported. Methods 1. Establish KC rat model and investigate the relationship between EMT and bladder fibrosis. Eighteen female Sprague-Dawley (SD) rats weighing 180-200g were randomly divided into three groups with 6 rats in each group. The control group, ketamine low-dose group (LK, 30mg/kg), ketamine high-dose group (HK, 60mg/kg) were injected intraperitoneally with ketamine solution or normal saline daily for 16 weeks. The number of unstable contractions, intravesical pressure (baseline, threshold, maximum), urinary output, maximum volume of the bladder, and bladder compliance were calculated. In the KC rat model, 24 female SD rats weighing 180-200 g were randomly divided into 4 groups, 6 rats in each group (control group) and 6 rats in ketamine (KET, 60mg/kg). Group B, ketamine + SB505124 (SB) group, abstinence group (abstinence group). SB group was given intraperitoneal injection of SB505124 (10 mg / kg) once a week on the basis of ketamine injection, abstinence group was given intraperitoneal injection of SB505124 (10 mg / kg) 12 weeks after ketamine injection for 4 weeks. SP1, E-cadherin-alpha-SMA co-expression. 3. Ketamine stimulated SV-HUC-1 cells for 72 hours by TGF-beta 1. Cell morphology was observed by phase contrast microscope. The expression of E-cadherin, FSP1, Vimentin, Fibronectin, alpha-SMA was detected by fluorescence quantitative PCR. The expression of E-cadherin, Fibronectin, alpha-SMA was detected by Western Blot assay. The expression of E-cadherin, Fibronectin and alpha-SMA was detected by fluorescence quantitative PCR at different time points after stimulation. The levels of TGF-beta 1 in the supernatant were measured. SB505124 blocked the TGF-beta 1 signaling pathway and Western Blot detected the induction of ketamine on EMT. The downstream signal molecules of TGF-beta 1 (Tbeta-R1, Tbeta-R2, Snail, Slug, Twist) were detected by fluorescence quantitative PCR. Results 1. After 16 weeks of ketamine exposure, 24-hour drinking and urine volume were measured in the control group, LK group, HK group. There was no significant difference between LK group and HK group. The number of urination in 6 hours was significantly increased in LK group and HK group, especially in HK group. The results of HE staining showed that compared with the control group, the epithelial layer of HK group became thinner and even the whole layer was missing, and the vascular dilatation was observed under the mucosa. The results showed that the level of E-cadherin was significantly lower in the experimental group, and the levels of Collagen I, Vimentin and Fibronectin were significantly higher in the HK group than in the LK group. The double immunofluorescence staining showed that E-cadherin+FSP1+cells were present in the bladder epithelium of the experimental group, suggesting the presence of EMT. Immunohistochemistry showed that TGF-beta 1 was expressed in the bladder epithelium of the LK and HK groups. After 16 weeks of drug intervention, 24-hour water intake and urine volume of rats in control group, KET group, SB group, abstinence group had no significant difference. Compared with KET group, the bladder maximal volume and bladder compliance were improved to some extent, but there was no significant difference between abstinence group and KET group. The results of HE staining and Mason staining showed that the bladder epithelial and interstitial fibrosis were improved in SB group compared with KET group. The expression of E-cadherin, Collagen I, Vimentin and Fibronectin was significantly decreased in the SB group compared with the KET group, suggesting that SB505124 could inhibit ketamine-induced EMT and could not significantly reverse ketamine-induced bladder damage after 4 weeks of withdrawal. 3. Ketamine (5-125 ug/ml) stimulated SV-HUC-1 cells for 24 h and 48 h, but no significant decrease in cell viability. Ketamine stimulated SV-HUC-1 cells for 72 h, and the cell morphology changed from cobblestone-like to spindle-like. QPCR showed that ketamine significantly down-regulated the expression of E-cadherin and up-regulated the expression of interstitial markers (FSP1, Vimentin, Fibronectin, alpha-SMA). ELISA showed that ketamine could up-regulate the expression of TGF-beta 1 in a time-and concentration-dependent manner. SB505124 significantly decreased the ability of ketamine to induce EMT. QPCR showed that ketamine enhanced the downstream signal molecule of TGF-beta 1 (Tbeta-SMA). Expression of R1, Tbeta-R2, Snail, Slug, Twist. Conclusion 1. Long-term ketamine abuse can lead to bladder dysfunction such as decreased bladder volume, unstable detrusor contraction, and decreased bladder compliance in rats. Pathologically, the bladder epithelial damage and interstitial fibrosis. 2. EMT phenomenon exists in ketamine-induced bladder fibrosis, and TGF-beta 1 specific blocker. SB505124 can inhibit EMT and interstitial fibrosis, EMT may be involved in bladder fibrosis. 3. Ketamine has no significant effect on the viability of human bladder epithelial cells in the dose range of 5-125 ug/ml. Ketamine can induce EMT in human bladder epithelial cells through TGF-beta 1 signaling pathway. Ketamine can up-regulate TGF-beta 1 expression in human bladder epithelial cells. Downstream signaling molecules (Snail, Slug, Twist) express.5.T beta R-1 may be a therapeutic target for KC fibrosis.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R694

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