本文選題：硫化氫 + 胃　； 參考：《山東大學(xué)》2009年碩士論文

【摘要】： 目的: 一氧化氮(NO)和一氧化碳(CO)屬于氣體遞質(zhì)(gaseous transmitter),參與多種生理功能的調(diào)節(jié)。硫化氫(Hydrogen Sulfide,H_2S)是一種無(wú)色具有臭雞蛋氣味的有毒氣體。有研究發(fā)現(xiàn)在人和大鼠的腦、血清、腎臟和肝臟中有相對(duì)高濃度的H_2S,這表明H_2S可能在這些組織中發(fā)揮生理作用,H_2S可能是除NO和CO外的第三種氣體遞質(zhì)。 機(jī)體可以通過(guò)胱硫醚β-合成酶(cystathionineβ-synthase,CBS)和胱硫醚γ-裂解酶(cystathionineγ-lyase,CSE)催化L-半胱氨酸(L-Cysteine)產(chǎn)生內(nèi)源性的H_2S。而硫氫化鈉(NaHS)作為H_2S的外源性供體已經(jīng)廣泛應(yīng)用于H_2S的研究當(dāng)中,因?yàn)樵谌芤褐兴軌蚍纸鉃镹a~+和HS~-,HS~-和H~+結(jié)合產(chǎn)生H_2S。有研究報(bào)道H_2S能夠引起腸道平滑肌的舒張,但是機(jī)制尚不確定。RudolfSchicho等發(fā)現(xiàn)在豚鼠和人回腸的腸神經(jīng)系統(tǒng)內(nèi)存在CSE,H_2S可能會(huì)通過(guò)腸神經(jīng)系統(tǒng)來(lái)調(diào)節(jié)胃腸道運(yùn)動(dòng),但內(nèi)源性H_2S是否調(diào)節(jié)胃的運(yùn)動(dòng)尚未見(jiàn)報(bào)道。本實(shí)驗(yàn)?zāi)康氖茄芯縃_2S對(duì)胃運(yùn)動(dòng)的影響及其可能機(jī)制。 方法: 實(shí)驗(yàn)選用健康雄性Wistar大鼠,實(shí)驗(yàn)前禁食12小時(shí),自由飲水。 手術(shù)操作和胃內(nèi)壓記錄 2%戊巴比妥鈉(56mg/kg)麻醉大鼠,依次行氣管插管、頸靜脈插管、股動(dòng)脈插管。將一塑料氣囊從食管插入胃腔后固定。氣囊內(nèi)注入生理鹽水使基礎(chǔ)胃內(nèi)壓維持于25mmHg。大鼠自發(fā)胃收縮活動(dòng)穩(wěn)定1個(gè)小時(shí)后,正式開(kāi)始實(shí)驗(yàn)。實(shí)驗(yàn)分2組:1)腹腔注射生理鹽水作為對(duì)照組;2)腹腔注射L-Cysteine(12mg/kg)或者NaHS組(5.6mg/kg)。 離體肌條制備和張力記錄 快速犧牲大鼠,取出胃放置于4℃Krebs液中。沿胃小彎剪開(kāi)胃腔,沖洗胃腔,再沿胃纖維走向剪取胃體縱行肌肌條(4mm×10mm)。將肌條分別放置在含有5ml krebs液(37℃)的浴槽中孵育,并每隔15分鐘更換一次浴槽中的krebs液。待肌條自發(fā)收縮穩(wěn)定后開(kāi)始實(shí)驗(yàn)。 統(tǒng)計(jì)學(xué)處理 在整體實(shí)驗(yàn)中,胃的運(yùn)動(dòng)強(qiáng)度用胃內(nèi)壓來(lái)表示,注射藥物前3分鐘的平均胃內(nèi)壓為基礎(chǔ)值(baseline),注射藥物后不同時(shí)間的平均胃內(nèi)壓為效應(yīng)值;在離體肌條實(shí)驗(yàn)中,肌條運(yùn)動(dòng)強(qiáng)度的大小用張力來(lái)衡量,加藥前3分鐘的平均張力胃基礎(chǔ)值(baseline),加藥后不同時(shí)間的平均張力為效應(yīng)值;效應(yīng)值與基礎(chǔ)值的比值為統(tǒng)計(jì)指標(biāo)R。 所有數(shù)據(jù)均采用均數(shù)±標(biāo)準(zhǔn)誤表示,采用單因素方差分析和f檢驗(yàn)進(jìn)行統(tǒng)計(jì)學(xué)處理,以P0.05為顯著性差異界值。 結(jié)果: 1.腹腔注射N(xiāo)aHS(5.6 mg/kg)和L-Cysteine(12 mg/kg)后,胃內(nèi)壓都迅速出現(xiàn)降低,然后逐漸恢復(fù)正常。 2.低濃度的NaHS(10~(-4)M)不影響胃體縱行肌肌條的收縮活動(dòng)。當(dāng)NaHS的濃度升高至5×10~(-4)M—10~(-3)M時(shí)胃體縱行肌肌條收縮活動(dòng)受到明顯抑制,其中5×10~(-4)M時(shí)抑制作用最強(qiáng),且在加入NaHS 10min時(shí)作用最強(qiáng),然后逐漸恢復(fù)。 3. ATP依賴(lài)性的K~+通道(K_(ATP))阻斷劑格列苯脲(glibenclamide)(10~(-7)M)能夠明顯逆轉(zhuǎn)NaHS(10~(-3)M)對(duì)胃體縱行肌肌條收縮活動(dòng)的抑制作用。但是,一氧化氮合酶非特異性阻斷劑N-硝基-L-精氨酸(N-nitro-L-arginine methylester,L-NAME)(10~(-4)M)和電壓依賴(lài)性的Na~+通道阻斷劑河豚毒素(tetrodotoxin,TTX)(10~(-6)M)對(duì)NaHS(10~(-3)M)的這種作用沒(méi)有影響。 4.低劑量的L-Cysteine(5×10~(-4)M)對(duì)胃體縱行肌肌條的收縮活動(dòng)沒(méi)有影響,當(dāng)劑量增加至(10~(-3) M—5×10~(-3) M)時(shí),胃體縱行肌肌條的收縮活動(dòng)受到明顯的劑量依賴(lài)性的抑制,該作用在加入L-Cysteine后1-2min時(shí)抑制作用最強(qiáng),10min內(nèi)恢復(fù)正常。 5.河豚毒素(tetrodotoxin,TTX)(10~(-6) M)和M受體阻斷劑阿托品(atropine)(10~(-6) M)都能部分阻斷L-Cysteine(10~(-3)M)對(duì)胃體縱行肌收縮活動(dòng)的抑制作用。但是,N-硝基-L-精氨酸(N-nitro-L-arginine methyl ester,L-NAME)(10~(-4)M)和格列苯脲(glibenclamide)(10~(-7)M)對(duì)L-Cysteine(10~(-3)M)的這種作用沒(méi)有影響。 6. CSE特異性抑制劑炔丙基甘氨酸(propargylglycine,PAG)(10~(-6)M-10~(-3)M)和CBS抑制劑氨基-羥乙酸(amino-oxyacetic acid,AOAA)(10~(-6)M-10~(-5)M)增強(qiáng)胃體縱行肌肌條收縮活動(dòng),且這種增強(qiáng)作用持續(xù)超過(guò)30min。 7. PAG(10~(-6)M)和AOAA(10~(-6)M)能夠部分阻斷L-Cysteine(10~(-3)M)對(duì)胃體縱行肌肌條收縮活動(dòng)的抑制,但是不影響NaHS(10~(-3)M)的作用。 結(jié)論: 1.外源性和內(nèi)源性H_2S均抑制胃的運(yùn)動(dòng)。 2.外源性H_2S對(duì)胃運(yùn)動(dòng)的抑制主要是通過(guò)誘導(dǎo)胃平滑肌細(xì)胞膜上的K_(ATP)開(kāi)放,而內(nèi)源性的H_2S的作用主要是通過(guò)腸神經(jīng)系統(tǒng)內(nèi)的膽堿能神經(jīng)實(shí)現(xiàn)的。 3.內(nèi)源性的H_2S對(duì)胃運(yùn)動(dòng)起著緊張性抑制作用。
[Abstract]:Objective:
Nitric oxide (NO) and carbon monoxide (CO) belong to the gas transmitter (gaseous transmitter) and are involved in a variety of physiological functions. Hydrogen sulfide (Hydrogen Sulfide, H_2S) is a colorless toxic gas with odorless egg odor. Studies have found that there is a relatively high concentration of H_2S in the brain, blood, kidney and liver of the human and rat, which indicates that H_2S can be found H_2S can be the third gas transmitter except NO and CO, which can play a physiological role in these tissues.
The organism can catalyze L- cysteine (L-Cysteine) to produce endogenous H_2S. with cysteine beta synthetase (cystathionine beta -synthase, CBS) and cystthioether gamma lyase (cystathionine gamma -lyase, CSE) as a H_2S exogenous donor, which has been widely used in H_2S studies because it can be decomposed in the solution. The combination of Na~+, HS~-, HS~- and H~+ combined with H_2S. has reported that H_2S can cause intestinal smooth muscle relaxation, but the mechanism is not yet determined that.RudolfSchicho and so on are found in the gut nervous system in guinea pigs and human ileum in CSE, H_2S may regulate gastrointestinal motility through the intestinal nervous system, but whether endogenous H_2S regulates gastric motility No report has been reported. The purpose of this experiment is to study the effect of H_2S on gastric motility and its possible mechanism.
Method:
The healthy male Wistar rats were randomly fasted for 12 hours before the experiment.
Surgical operation and intragastric pressure recording
2% pentobarbital sodium (56mg/kg) rats were anesthetized with tracheal intubation, jugular intubation, and femoral artery intubation. A plastic balloon was inserted into the stomach cavity from the esophagus. The air bag was injected with saline to maintain the underlying gastric pressure in the spontaneous gastric contractile activity of 25mmHg. rats. The experiment was formally started. The experiment was divided into 2 groups: 1) intraperitoneal injection. Normal saline was used as control group; 2) intraperitoneal injection of L-Cysteine (12mg/kg) or NaHS group (5.6mg/kg).
Preparation of isolated muscle strips and record of tension
The rats were sacrificed quickly and placed in the Krebs solution at 4 degrees centigrade. The gastric cavity was cut along the gastric small bend, the stomach cavity was washed, and then the muscle strips of the stomach were cut along the gastric fibers (4mm x 10mm). The muscle strips were incubated in the bath containing 5ml Krebs liquid (37 degrees C), and the Krebs solution in the bath was changed every 15 minutes. After the muscle strips were spontaneously contracted, the muscle strips were stable and stable. Start the experiment.
Statistical treatment
In the whole experiment, the exercise intensity of the stomach was expressed by intragastric pressure. The average intragastric pressure of 3 minutes before injection was the base value (baseline). The average intragastric pressure was the effect value at different time after injection. In the experiment of isolated muscle strips, the intensity of muscle strips was measured by tension, and the average tension of gastric base was 3 minutes before adding medicine (base Line) the average tension at different times after adding medicine is the effect value; the ratio of the effect value to the base value is the statistical index R..
All data were expressed by means of mean + standard error, and one-way ANOVA and F test were used for statistical analysis. P0.05 was a significant difference value.
Result:
1. intraperitoneal injection of NaHS (5.6 mg/kg) and L-Cysteine (12 mg/kg) resulted in a rapid decrease in intragastric pressure and gradually returned to normal.
2. the low concentration of NaHS (10~ (-4) M) did not affect the contractile activity of the longitudinal muscle strips of the stomach. When the concentration of NaHS increased to 5 * 10~ (-4) M 10~ (-3) M, the contractile activity of the longitudinal muscle strips of the stomach was obviously inhibited, and the inhibitory effect was strongest when 5 * 10~ (10~) was the strongest, and then gradually recovered.
3. ATP dependent K~+ channel (K_ (ATP)) blocker glibenclamide (glibenclamide) (10~ (-7) M) can obviously reverse the inhibition of NaHS (10~ (-3) M) on the contractile activity of the longitudinal muscle strips of the stomach. However, the nitric oxide synthase nonspecific blocking agent, nitroedible arginine, and voltage dependence The Na~+ channel blocker, tetrodotoxin (TTX) (10~ (-6) M), has no effect on NaHS (10~ (-3) M).
4. the low dose of L-Cysteine (5 x 10~ (-4) M) had no effect on the contractile activity of the longitudinal muscle strips of the gastric body. When the dose increased to (10~ (-3) M - 5 * 10~ (-3) M), the contraction activity of the muscle strips of the gastric body was obviously dose-dependent, and the inhibitory effect was strongest in L-Cysteine 1-2min.
5. tetrodotoxin (TTX) (10~ (-6) M) and M receptor blocking agent atropine (atropine) (10~ (-6) M) can partially block the inhibitory effect of L-Cysteine (10~) on the contractile activity of the longitudinal muscle of the stomach. The effect of ysteine (10~ (-3) M) is not affected.
The 6. CSE specific inhibitor propargylglycine (PAG) (10~ (-6) M-10~ (-3) M) and the CBS inhibitor amino hydroxyacetic acid (AMINO-OXYACETIC acid, AOAA) enhanced the contractile activity of the longitudinal muscle strips of the stomach body, and this enhancement continued to exceed that of the muscle.
7. PAG (10~ (-6) M) and AOAA (10~ (-6) M) can partially block the inhibitory activity of L-Cysteine (10~ (-3) M) on the contractile activity of the longitudinal muscle strips of the stomach body, but it does not affect the effect.
Conclusion:
1. both exogenous and endogenous H_2S inhibit the motion of the stomach.
2. the inhibitory effect of exogenous H_2S on gastric motility is mainly through the induction of K_ (ATP) on the membrane of the smooth muscle cells of the stomach, and the role of endogenous H_2S is realized mainly through the cholinergic nerve in the intestinal nervous system.
3. endogenous H_2S plays a tonic inhibitory role on gastric motility.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R333

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