【摘要】：背景長期濫用毒品氯胺酮(ketamine)會引發(fā)膀胱間質(zhì)炎癥(ketamine-induced cystitis,KC),主要臨床表現(xiàn)為尿頻、尿急、尿痛、排尿困難及急迫性尿失禁等。膀胱纖維化是中晚期KC患者的嚴(yán)重并發(fā)癥。膀胱壁由于纖維結(jié)締組織積聚而發(fā)生重塑,引起膀胱纖維化、攣縮,使其變?yōu)樾∪萘�、高壓力的低順�?yīng)性膀胱,導(dǎo)致膀胱貯尿和排尿功能障礙,進(jìn)一步發(fā)展會導(dǎo)致上尿路積水和腎功能損害,危及生命。目前少有研究涉及ketamine引發(fā)膀胱纖維化的機(jī)制。上皮-間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transitions,EMT)是指上皮細(xì)胞失去極性、在形態(tài)學(xué)上向間質(zhì)細(xì)胞轉(zhuǎn)變的生物學(xué)過程。目前已證實(shí)EMT廣泛參與腎臟、肝臟、腸壁及肺臟的纖維化,并已成為有效終止、逆轉(zhuǎn)臟器纖維化的重要靶點(diǎn),然而其在膀胱纖維化中的作用尚未見報(bào)道。本項(xiàng)目擬從EMT介導(dǎo)器官纖維化入手,結(jié)合體內(nèi)外模型,通過分子機(jī)制研究闡明TGF-β1信號通路介導(dǎo)的EMT在KC纖維化發(fā)生發(fā)展中的機(jī)制。方法1.建立KC大鼠模型并探討EMT與膀胱纖維化的關(guān)系雌性Sprague-Dawley(SD)大鼠18只,體重180-200g,隨機(jī)分為3組,每組6只,分別為對照組(Control),ketamine低劑量組(LK,30mg/kg),ketamine高劑量組(HK,60mg/kg)。每日腹腔注射ketamine溶液或生理鹽水,持續(xù)16周。采用代謝籠記錄24h飲水量和尿量。采用排尿印跡法評估尿頻情況。對大鼠行尿動力檢查,記錄排尿收縮次數(shù)、逼尿肌不穩(wěn)定收縮次數(shù)、膀胱內(nèi)壓力(基線值,閾值,最大值)、排尿量、膀胱最大容量,計(jì)算膀胱順應(yīng)性。處死大鼠后取膀胱組織,制作石蠟切片,HE及Masson染色評估膀胱上皮損害及間質(zhì)纖維化情況,免疫組化實(shí)驗(yàn)評估 E-cadherin、Collageon I、Vimentin、Fibronectin 表達(dá)情況,E-cadherin-FSPl免疫熒光共定位評估膀胱上皮EMT發(fā)生情況。2.在KC大鼠模型中,明確TGF-β1特異性阻斷劑SB505124對EMT及膀胱纖維化的調(diào)節(jié)作用雌性SD大鼠24只,體重180-200g,隨機(jī)分為4組,每組6只,分別為對照組(control),ketamine(KET,60mg/kg)組,ketamine+SB505124(SB)組,戒斷組(abstinence組)。SB組在注射ketamine的基礎(chǔ)上每周腹腔注射SB505124(10 mg/kg)一次,abstinence組注射ketamine后12周戒斷4周。排尿印記、尿動力檢查、HE染色、Masson染色、免疫組化分析同第一部分,免疫熒光雙標(biāo)檢測 E-cadherin-FSP1、E-cadherin-α-SMA 共表達(dá)情況。3.Ketamine通過TGF-β1調(diào)節(jié)人膀胱上皮細(xì)胞EMT的分子機(jī)制研究培養(yǎng)人永生化膀胱上皮(SV-HUC-1)細(xì)胞,CCK-8法檢測ketamine(5-125μg/ml)對細(xì)胞活力的影響。Ketamine(125 μg/ml)刺激 SV-HUC-1 細(xì)胞 72h,相差顯微鏡觀察細(xì)胞形態(tài)變化。Ketamine(125 μg/ml)刺激細(xì)胞24h,熒光定量 PCR 法檢測 EMT 標(biāo)志物(E-cadherin、FSP1、Vimentin、Fibronectin、α-SMA)表達(dá)。Ketamine(5 to 125μg/mL)刺激細(xì)胞 48h,Western Blot 檢測 E-cadherin、Fibronectin、α-SMA 蛋白表達(dá)水平。Ketamine(5 to 125μg/mL)刺激細(xì)胞不同時(shí)間,ELISA檢測上清液TGF-β1水平變化。SB505124阻斷TGF-β1信號通路,Western Blot檢測ketamine對EMT的誘導(dǎo)作用。熒光定量PCR法檢測TGF-β1 下游信號分子(Tβ-R1,Tβ-R2,Snail,Slug,Twist)。結(jié)果1.大鼠經(jīng)過16周ketamine暴露后,24小時(shí)飲水量和尿量在control組、LK組、HK組間無明顯差異。LK組和HK組6小時(shí)排尿次數(shù)較對照組顯著增多,HK組更為明顯。尿動力學(xué)結(jié)果顯示,長期濫用ketamine后,大鼠的排尿頻率、逼尿肌無抑制收縮次數(shù)、膀胱最大壓力均明顯升高,而膀胱最大容量和膀胱順應(yīng)性明顯降低,且隨接ketamine劑量的增加而更加嚴(yán)重。HE染色結(jié)果顯示:與control組相比,LK組、HK組上皮層明顯變薄甚至全層缺失形成潰瘍,黏膜下見不同程度的血管擴(kuò)張。Masson染色結(jié)果顯示:與control組相比,LK組、HK組膀胱肌層中存在大量膠原纖維,HK組更加嚴(yán)重。免疫組化結(jié)果證實(shí)實(shí)驗(yàn)組 E-cadherin 水平明顯降低,Collagen I、Vimentin 和 Fibronectin 蛋白明顯升高,且HK組較LK組變化更加明顯。免疫熒光雙標(biāo)結(jié)果顯示,實(shí)驗(yàn)組膀胱上皮層出現(xiàn)E-cadherin+FSP1+細(xì)胞,提示EMT的存在。免疫組化示TGF-β1在LK和HK組膀胱上皮表達(dá)顯著升高。2.大鼠經(jīng)過16周藥物干預(yù)后,24小時(shí)飲水量和尿量在control組、KET組、SB組、abstinence組無明顯差異。與KET組相比,SB組排尿次數(shù)顯著降低,而abstinence組無明顯變化。SB505124干預(yù)后,大鼠的排尿頻率、逼尿肌無抑制收縮次數(shù)、膀胱最大壓力較KET組均明顯降低,膀胱最大容量和膀胱順應(yīng)性也得到不同程度的改善,而abstinence組這些指標(biāo)與KET組無明顯差異。HE染色和Masson染色結(jié)果顯示:與KET組相比,SB組膀胱上皮及間質(zhì)纖維化均得到改善。免疫組化結(jié)果表明,SB505124可以顯著抑制ketamine對E-cadherin、Collagen I、Vimentin和Fibronectin蛋白表達(dá)的影響。免疫熒光雙標(biāo)實(shí)驗(yàn)顯示:與 KET 組相比,SB 組 E-cadherin+FSP1+細(xì)胞和 E-cadherin+α-SMA+細(xì)胞均明顯減少,提示SB505124可以抑制ketamine誘導(dǎo)EMT。戒斷4周不能明顯逆轉(zhuǎn)ketamine造成的膀胱損害。3.Ketamine(5-125μg/ml)刺激 SV-HUC-1 細(xì)胞 24h 和 48h,細(xì)胞活力無明顯降低。Ketamine刺激SV-HUC-1細(xì)胞72 h,細(xì)胞形態(tài)由典型的鵝卵石樣轉(zhuǎn)變?yōu)榧忓N樣。qPCR顯示ketamine顯著下調(diào)E-cadherin表達(dá),上調(diào)間質(zhì)標(biāo)志物(FSP1、Vimentin、Fibronectin、α-SMA)的表達(dá)。Western Blot 驗(yàn)證 ketamine降低E-cadherin蛋白水平,上調(diào)Fibronectin、α-SMA蛋白水平,具有濃度依賴效應(yīng)。ELISA實(shí)驗(yàn)表明,ketamine可以上調(diào)TGF-β1表達(dá),具有時(shí)間和濃度依賴效應(yīng)。SB505124明顯降低ketamine誘導(dǎo)EMT的能力。qPCR顯示ketamine增強(qiáng) TGF-β1 下游信號分子(Tβ-R1,Tβ-R2,Snail,Slug,Twist)的表達(dá)。結(jié)論1.長期濫用氯胺酮可以導(dǎo)致大鼠膀胱容量降低、逼尿肌不穩(wěn)定收縮、膀胱順應(yīng)性降低等膀胱功能障礙,病理上表現(xiàn)為膀胱上皮破壞和間質(zhì)纖維化。2.氯胺酮性膀胱纖維化中存在EMT現(xiàn)象,TGF-β1特異性阻斷劑SB505124可以抑制EMT和間質(zhì)纖維化,EMT可能參與膀胱纖維化。3.氯胺酮在5-125 μg/ml劑量范圍對人膀胱上皮細(xì)胞的活力無明顯影響。氯胺酮可以通過TGF-β1信號通路誘導(dǎo)人膀胱上皮細(xì)胞發(fā)生EMT。4.氯胺酮刺激人膀胱上皮細(xì)胞后,可以上調(diào)TGF-β1下游信號分子(Snail,Slug,Twist)表達(dá)。5.TβR-1可能是KC纖維化的治療靶點(diǎn)。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R694

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本文編號：2198060