本文關(guān)鍵詞： 硫化氫 腎動脈 CSE ATP敏感鉀通道 格列苯脲 Bay K8644　出處：《河北醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 硫化氫(hydrogen sulfide,H2S)一直被認(rèn)為是一種無色、具有臭雞蛋味的毒性氣體,它可以抑制細(xì)胞色素氧化酶進(jìn)而抑制線粒體的呼吸作用。H2S是繼內(nèi)源性氣體分子一氧化氮(nitric oxide,NO)、一氧化碳(carbon monoxide,CO)后發(fā)現(xiàn)的第三個內(nèi)源性氣體信使(Gasotransmitter)。已證實(shí)H2S可以調(diào)節(jié)生理及病理狀態(tài)下心臟的功能,并在自發(fā)性高血壓大鼠、低氧性肺動脈高血壓大鼠、高肺血流量所致肺動脈高壓及腎血管性高血壓的發(fā)病中也起著重要的調(diào)節(jié)作用。H2S可通過開放KATP通道使容量血管大鼠胸主動脈舒張,還可通過KATP通道和內(nèi)依賴性超極化因子(endothelium-derived hyperpolarizing factor,EDHF)介導(dǎo)的KCa通道共同作用使阻力血管腸系膜動脈舒張。可見H2S誘導(dǎo)的舒血管效應(yīng)有別于其他內(nèi)源性氣體信號分子(如NO、CO是通過鳥苷酸環(huán)化酶cGMP通路起作用)。H2S還可以抑制血管平滑肌細(xì)胞的增殖和促進(jìn)其凋亡、緩解血管結(jié)構(gòu)的重建。提示H2S可能在心血管系統(tǒng)穩(wěn)態(tài)調(diào)節(jié)中發(fā)揮著重要作用。腎動脈張力是調(diào)節(jié)血管外周阻力的重要因素,腎血管阻力增高是高血壓病時腎血流動力學(xué)改變的重要特征。腎動脈的病變導(dǎo)致管腔的狹窄進(jìn)而腎臟缺血刺激腎臟皮質(zhì)內(nèi)球旁裝置細(xì)胞分泌腎素過度,引發(fā)腎素血管緊張素系統(tǒng)過度激活,全身小動脈收縮血壓升高,導(dǎo)致腎血管性高血壓。可見腎動脈的張力在高血壓病和腎血管性高血壓的發(fā)病過程中起重要作用,而H2S對它的影響至今尚無人報道。 目的 應(yīng)用離體血管環(huán)張力測定技術(shù)觀察H2S對家兔離體腎動脈血管環(huán)張力的調(diào)節(jié)作用,探討其可能的作用機(jī)制。 方法 1.測定正常家兔血漿中H2S的含量。 在試管中加入1%(質(zhì)量分?jǐn)?shù))醋酸鋅0.5 ml,蒸餾水2.5 ml,血漿0.1 ml,混勻。然后加入7.2 mol/L鹽酸(含20 mmol/L N,N-二甲基-對苯二胺鹽酸鹽)0.5 ml,再加入1.2 mol/L HCl(含30 mmol/L FeCl3)0.4 ml,室溫孵育20 min,然后加入10%三氯醋酸1 ml。將上述試管內(nèi)容物離心5 min后取上清,用分光光度計(jì)在665 nm波長處檢測光吸收度。用H2S標(biāo)準(zhǔn)曲線計(jì)算血漿H2S濃度,結(jié)果用μmol/L表示。 2.測定腎動脈上硫化氫生成酶(cystathionine -lyase,CSE)的活性。 取家兔的腎動脈在冰冷的磷酸鉀緩沖液(50 mmol/L,pH 6.8)中研磨成勻漿。反應(yīng)在25 ml錐形瓶中進(jìn)行。反應(yīng)體積1 ml,含磷酸鉀緩沖液(100 mmol/L,pH 7.4)、L-半胱氨酸(10 mmol/L)、5’-磷酸吡哆醛(2 mmol/L)和10%(V/V)組織勻漿。在中央室中加入10%(W/W)醋酸鋅0.5 ml,并放入濾紙?jiān)黾游彰娣e。用N2將燒瓶充盈30 s后,石蠟?zāi)し饪?轉(zhuǎn)移到37℃水浴搖床中開始反應(yīng),90 min后向其中注入50%(W/W)三氯醋酸0.5 ml中止反應(yīng),繼續(xù)水浴60 min。將中央室的內(nèi)容物轉(zhuǎn)移到含3.5 ml蒸餾水的試管中,加入20 mmol/L對苯二胺鹽酸鹽0.5 ml和30 mmol/L FeCl3 0.4 ml,20 min后用分光光度計(jì)在665 nm波長處檢測光吸收度。用H2S標(biāo)準(zhǔn)曲線計(jì)算溶液中H2S含量,H2S產(chǎn)出率由(pmol/mg protein/minute)來表示,反映CSE的活性。 3.硫化氫對離體家兔腎動脈血管環(huán)張力的調(diào)節(jié)。 運(yùn)用離體血管環(huán)灌流系統(tǒng),觀察硫化氫作用后血管環(huán)張力的變化,由RM6240生物信號采集處理系統(tǒng)進(jìn)行記錄分析,檢測血管環(huán)張力的變化并探討其可能的機(jī)制。 結(jié)果 1.正常兔血漿中H2S的含量為(44.39±4.9)μmol/L。 2.正常家兔腎動脈上H2S生成率為(143.94±4.80)pmol/mg protein/minute。 3. (1)外源性的H2S(NaHS 50,100,200,400,800μmol/L)可以劑量依賴性地舒張由KCl(50 mmol/L)預(yù)收縮的腎動脈血管環(huán),其濃度反應(yīng)曲線的IC50值為281.46±17.26μmol/L,最大舒張率為89.97±2.40%。(2)用KATP通道阻斷劑格列苯脲(glibenclamide, 20μmol/L)、鈣通道的開放劑Bay K8644(500 nmol/L)、NO合酶的抑制劑L-NAME(100μmol/L)和環(huán)氧合酶阻斷劑吲哚美辛(10μmol/L)預(yù)處理以及去除血管內(nèi)皮后,H2S的舒張效應(yīng)均被顯著抑制,濃度反應(yīng)曲線均明顯右移,其IC50值分別增大為(299.69±8.64μmol/L,P 0.05)、(405.32±2.84μmol/L,P 0.05)、(369.25±0.85μmol/L,P 0.05)、(387.18±26.26μmol/L,P 0.01)和(331.15±4.13μmol/L,P 0.05)。且格列苯脲、Bay K8644和去除血管內(nèi)皮后使最大舒張率分別減小為(68.65±4.31%,P 0.01)、(80.10±3.11%,P 0.05)和(76.03±2.35%,P 0.01),但應(yīng)用L-NAME和吲哚美辛并沒有改變H2S的最大舒張作用。(3)預(yù)先給予鳥苷酸環(huán)化酶的抑制劑ODQ(10μmol/L)以及EDHF的抑制劑apamin(50 nmol/L)和charybdotoxin(50 nmol/L)預(yù)處理后,對H2S的舒張作用沒有顯著改變,其IC50值分別為(295.31±16.07μmol/L,P 0.05)和(261.47±36.57μmol/L,P 0.05)。(4)做KCl(5,15,25,35,45,55,65 mmol/L)收縮腎動脈環(huán)的量效曲線,其IC50值為14.91±0.16 mmol/L。(5)先給予H2S合酶的抑制劑PPG預(yù)處理后,KCl的濃度量效曲線左移,其IC50值減小為(11.65±1.08 mmol/L,P 0.05)。(6)先給予H2S(NaHS 200μmol/L)預(yù)處理后,KCl的濃度量效曲線右移,其IC50值增大為(19.16±1.09 mmol/L,P 0.05)。 結(jié)論 1.正常家兔血漿中含有H2S。 2.正常家兔腎動脈含有CSE,且能產(chǎn)生H2S。 3.實(shí)驗(yàn)數(shù)據(jù)表明H2S對腎動脈有舒張作用,此作用是由直接開放血管平滑肌上的KATP通道,同時關(guān)閉鈣通道來實(shí)現(xiàn)的;且此作用是內(nèi)皮依賴型的,與一氧化氮和前列環(huán)素有協(xié)同舒張作用,但與鳥苷酸環(huán)化酶途徑和EDHF途徑無關(guān);血管組織生成的內(nèi)源性H2S可拮抗KCl引起的血管收縮作用。
[Abstract]:Hydrogen sulfide (hydrogen sulfide H2S) has been considered as a colorless, toxic gas with a rotten egg smell, it can inhibit the respiration of.H2S and inhibition of the mitochondrial cytochrome oxidase is the endogenous nitric oxide gas molecules (nitric oxide, NO), carbon monoxide (carbon monoxide, CO) third endogenous gaseous messenger after the discovery (Gasotransmitter). It has been confirmed that H2S can regulate the heart function in physiological and pathological conditions, and in spontaneously hypertensive rats, hypoxic pulmonary hypertension in rats, the incidence of high pulmonary blood flow induced pulmonary hypertension and renal vascular hypertension also plays an important role in the regulation of.H2S by opening KATP the channel capacity of vessels of rat thoracic aorta, but also dependent hyperpolarizing factor through the KATP channel (endothelium-derived hyperpolarizing factor, and EDHF) KCa channel mediated interaction With the relaxation of mesenteric artery vascular resistance. The vasodilatory effect induced by visible H2S is different from other gasotransmitter (such as NO, CO is the guanylate cyclase cGMP pathway).H2S can also inhibit the proliferation of vascular smooth muscle cells and promote its apoptosis, relieve the reconstruction of vascular structure. May indicate that H2S play an important role in the regulation of cardiovascular homeostasis. Renal artery tension is an important factor in the regulation of peripheral vascular resistance, increased renal vascular resistance is an important feature of renal hemodynamics in hypertensive patients. Renal artery lesions lead to luminal stenosis of the renal cortex and renal ischemia stimulate the juxtaglomerular apparatus cells renin secretion excessive, cause renin angiotensin system activation, systemic arterial systolic blood pressure increased, resulting in renal vascular hypertension. Visible renal artery tension in hypertension and renal blood vessels The pathogenesis of high blood pressure plays an important role, and the impact of H2S on it has yet to be reported.
objective
To observe the effect of H2S on the tension of the isolated renal artery ring of rabbit in vitro, the possible mechanism of its possible action was investigated.
Method
1. the content of H2S in normal rabbit plasma was measured.
1% in vitro (mass fraction) of zinc acetate 0.5 ml distilled water, 2.5 ml, 0.1 ml plasma, then mix. Add 7.2 mol/L of hydrochloric acid (containing 20 mmol/L N, N- two - two of benzene methyl amine hydrochloride) 0.5 ml, adding 1.2 mol/L HCl (30 mmol/L FeCl3) 0.4 ml. After incubation at room temperature for 20 min, then adding 10% three 1 ml. the chloroacetic acid content after 5 min centrifuge tube Torikami Kiyo, in the detection wavelength of 665 nm optical absorption spectrophotometer. Calculation of plasma H2S concentration by H2S standard curve, the results were expressed as mol/L.
2. the activity of cystathionine -lyase (CSE) was measured on the renal artery.
鍙栧鍏旂殑鑲懼姩鑴夊湪鍐板喎鐨勭７閰擱捑緙撳啿娑，

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