劉書(shū)苑,陳 科,劉 敏,何紅暉,謝艷紅,謝曉云,莫朝暉
(中南大學(xué)湘雅三醫(yī)院內(nèi)分泌科,湖南長(zhǎng)沙410013)
鎢酸鈉促進(jìn)新生豬胰島細(xì)胞體外增殖、分化及分泌功能
劉書(shū)苑#,陳 科,劉 敏,何紅暉,謝艷紅,謝曉云,莫朝暉*
(中南大學(xué)湘雅三醫(yī)院內(nèi)分泌科,湖南長(zhǎng)沙410013)
目的 觀(guān)察鎢酸鈉在體外對(duì)新生豬胰島細(xì)胞增殖及功能活性的影響。方法 分離和純化新生豬胰島細(xì)胞后,與鎢酸鈉共育,隔天進(jìn)行細(xì)胞計(jì)數(shù)、MTT實(shí)驗(yàn),取最佳濃度300 μmol/L鎢酸鈉與細(xì)胞共同培養(yǎng)3d,收集細(xì)胞進(jìn)行葡萄糖刺激實(shí)驗(yàn),并分別用Western blot、RT-PCR方法檢測(cè)細(xì)胞內(nèi)胰島素蛋白(INSULIN)、細(xì)胞增殖核抗原(PCNA)和胰十二指腸同源框蛋-1(PDX-1)mRNA、葡萄糖轉(zhuǎn)運(yùn)子-2(GLUT-2)mRNA的表達(dá)。結(jié)果 300 μmol/L鎢酸鈉處理后胰島細(xì)胞增殖活性及葡萄糖刺激實(shí)驗(yàn)中胰島素分泌明顯高于對(duì)照組(P<0.05),鎢酸鈉干預(yù)組PCNA蛋白和INSULIN蛋白表達(dá)分別為2.24±0.19和1.62±0.17,顯著高于對(duì)照組的1.17±0.13和0.37±0.08(P<0.05,P<0.01);PDX-1和GLUT-2mRNA表達(dá)分別為0.34±0.05和1.06±0.10,顯著高于對(duì)照組的0.11±0.03和0.13±0.02(P<0.01)。結(jié)論 低濃度(300 μmol/L)鎢酸鈉具有促進(jìn)新生豬胰島細(xì)胞增殖分化、增強(qiáng)胰島細(xì)胞功能、促進(jìn)胰島素分泌的作用。
鎢酸鈉;新生豬胰島細(xì)胞;增殖和分化;胰島功能
鎢酸鈉含有微量元素鎢,具有很強(qiáng)的降糖作用,可將1型及2型糖尿病動(dòng)物模型的血糖降至正常水平而不引起低血糖[1-2]。盡管鎢酸鈉與釩酸鈉的生物學(xué)性質(zhì)相似,毒性卻明顯較釩酸鈉低,具有相對(duì)較高的生物利用度,這使得它在糖尿病防治方面較其他微量元素更具前景性。近年來(lái)發(fā)現(xiàn)其作用機(jī)制與釩酸鈉有所差異,除具有類(lèi)胰島素作用外,還具有促進(jìn)胰島素分泌和促進(jìn)胰島細(xì)胞再生作用,因而即使撤藥后相當(dāng)長(zhǎng)一段時(shí)間,治療組動(dòng)物血糖水平仍保持正?;虻陀趯?duì)照組[1,3]。但迄今所進(jìn)行的研究都是在小動(dòng)物,基本為鼠類(lèi),而且這方面的研究結(jié)果也有分歧。
由于新生豬胰島具有較高的增殖及生長(zhǎng)成熟的潛能,不僅能較容易地在體外大量培養(yǎng),而且相對(duì)于胎豬胰島細(xì)胞具有對(duì)糖刺激的良好反應(yīng)性,成為異種胰島細(xì)胞移植最有前途的供體來(lái)源。但由于未成熟,移植后需要一段時(shí)間才能控制血糖,如何在體外培養(yǎng)過(guò)程中促進(jìn)新生豬胰島細(xì)胞增殖分化成熟與增強(qiáng)胰島細(xì)胞功能,也被人們所關(guān)注。本研究探討了鎢酸鈉降糖作用機(jī)制,也為改善新生豬胰島細(xì)胞質(zhì)量提高胰島移植效果提供新的可能藥物。
Ⅴ型膠原酶(Sigma公司);真胰島素檢測(cè)試劑盒(北京泰格科信生物科技有限公司);RNA抽提試劑盒(Gentre公司);PCNA多克隆抗體(NO:13-3900)、Insulin蛋白多克隆抗體(Zeymed公司);辣根過(guò)氧化物酶標(biāo)記抗鼠二抗、羊抗鼠actin、辣根過(guò)氧化物酶標(biāo)記抗羊二抗、ECL(Santa Cruz公司)。
出生3~5 d新生豬4頭(湘雅醫(yī)學(xué)院動(dòng)物實(shí)驗(yàn)部,質(zhì)量1.2~1.5 kg)氯胺酮麻醉后,無(wú)菌狀態(tài)下剖腹取胰,去除肉眼所見(jiàn)的胰腺外組織、導(dǎo)管、結(jié)締組織和淋巴組織、胰腺被膜后,將胰腺剪碎;加入0.5 g/L濃度的Ⅴ型膠原酶30 mL錐形瓶中37℃水浴劇烈振蕩10 mm。用4℃ D-Hank's液中止消化。于4℃,1 000 r/min離心1 min,棄上清液,加入含20%小牛血清、青霉素100 U/mL、鏈霉素100 U/mL、谷氨酰氨1%增補(bǔ)RPMI1640液,置放于37℃、5%CO2和95%空氣的培養(yǎng)箱內(nèi)培養(yǎng),每個(gè)培養(yǎng)瓶?jī)?nèi)約1×105個(gè)細(xì)胞,實(shí)驗(yàn)備用。收集小部分胰島細(xì)胞在倒置顯微鏡下觀(guān)察細(xì)胞形態(tài)。雙硫腙(DTZ)染色鑒定胰島細(xì)胞純度在80%以上,臺(tái)盼藍(lán)染色計(jì)數(shù)細(xì)胞存活率為95%。
配制2×104細(xì)胞懸液接種于 24孔板,每孔1 mL。分別于接種的第1、3、5和7天對(duì)鎢酸鈉濃度分別為 50、150、300、500 和700 μmol/L的 5 個(gè)實(shí)驗(yàn)組及對(duì)照組進(jìn)行細(xì)胞計(jì)數(shù)并取平均值,作出細(xì)胞生長(zhǎng)曲線(xiàn)。
根據(jù)此實(shí)驗(yàn)結(jié)果取300 μmol/L鎢酸鈉處理組為實(shí)驗(yàn)組進(jìn)行以下實(shí)驗(yàn)。
配制1×105/mL細(xì)胞懸液接種于96孔板,設(shè)實(shí)驗(yàn)組(300 μmol/L鎢酸鈉干預(yù))和對(duì)照組各8個(gè)復(fù)孔,每孔200 μL細(xì)胞懸液,共接種4塊板。分別于第1、3、5 和7 天取1 塊96 孔板加入5 g/L MTT 20 μL,37℃培養(yǎng)箱孵育4 h后,棄上清液,每孔加150 μL DMSO振蕩10 mm后,在全自動(dòng)酶標(biāo)儀上用550 nm波長(zhǎng)比色,記錄每孔A值并計(jì)算出每天各組的A均值,制表作圖,批間變異系數(shù)8.1%,批內(nèi)變異系數(shù)2.2%。
300 μmol/L濃度鎢酸鈉干預(yù)3 d后實(shí)驗(yàn)組和對(duì)照組用D-Hank's液洗滌后,均分成5份。分別先后用Hanks液配制的含2.7 mmol/L的低糖培養(yǎng)基和26.7 mmol/L的高糖培養(yǎng)基置換原培養(yǎng)基后孵育4 h,收集上清液,檢測(cè)真胰島素含量,批間變異系數(shù)12.1%,批內(nèi)變異系數(shù)3.2%,并計(jì)算胰島素刺激指數(shù)。
干預(yù)3 d后分別收集實(shí)驗(yàn)組和對(duì)照組胰島細(xì)胞按Gentre RNA試劑盒抽提細(xì)胞總 RNA,取1 μg總RNA以O(shè)ligo(DT)18為引物進(jìn)行反轉(zhuǎn)錄,反轉(zhuǎn)錄體系20 μL,將反轉(zhuǎn)錄的產(chǎn)物用于PCR擴(kuò)增,引物設(shè)計(jì)見(jiàn)表1。PCR反應(yīng)按以下條件進(jìn)行:94℃預(yù)變性2.5 min,94℃變性30 s,PDX-1按52℃復(fù)性30 s;GLUT-2按58℃復(fù)性30 s;72℃延伸1 min。共擴(kuò)增34個(gè)循環(huán),2%的瓊脂糖凝膠電泳PCR產(chǎn)物,凝膠成像系統(tǒng)顯影拍照,Gene Tool分析軟件測(cè)定吸光度值,并計(jì)算目的基因與β-actin吸光度比值。
表1 目的基因引物序列及擴(kuò)增產(chǎn)物Table 1 Target gene's primer sequences and amplif ied product
用細(xì)胞裂解緩沖液(50 mmol/L Tris-Hcl(pH 8.0),150 mmol/L nacl,1% Triton X-100,0.2%NaN3,0.5% 去氧膽酸鈉,10 mg/L Aprotinnin,1 mg/L PMSF)裂解細(xì)胞,抽提鎢酸鈉干預(yù)和對(duì)照組新生豬胰島樣細(xì)胞總蛋白。Bradford法測(cè)蛋白含量后,置 -70℃冰箱保存。取40 μg細(xì)胞總蛋白于10%SDS-PAGE膠中電泳,電轉(zhuǎn)移至PVDF膜上。含5%脫脂奶粉的PBS封閉1 h,用鼠抗PCNA多克隆抗體(1∶500稀釋)的 PBS,4℃溫育過(guò)夜,PBS洗膜5 min×3次,用辣根過(guò)氧化物酶標(biāo)記抗鼠二抗(1∶2 000稀釋)的 PBS溫育1 h,洗膜后,ECL 發(fā)光自顯影,洗片顯帶。同膜洗脫后,用羊抗鼠 actin(1∶1 000稀釋)一抗,辣根過(guò)氧化物酶標(biāo)記抗羊二抗(1∶2 000稀釋)重新雜膜,發(fā)光自顯影,洗片顯帶作為內(nèi)對(duì)照。所有雜交信號(hào)在成像分析儀系統(tǒng)測(cè)定條帶密度。目的條帶水平以PCNA/actin的比值表示。同法檢測(cè)Insulin蛋白。
各實(shí)驗(yàn)獨(dú)立重復(fù)3次以上。實(shí)驗(yàn)數(shù)據(jù)采用均數(shù)±標(biāo)準(zhǔn)差(±s)表示,統(tǒng)計(jì)學(xué)處理采用SPSS 11.0統(tǒng)計(jì)軟件。兩樣本均數(shù)比較采用兩獨(dú)立樣本t檢驗(yàn),組間比較用方差分析。計(jì)數(shù)資料采用卡方檢驗(yàn)。
剛分離的新生豬胰島細(xì)胞團(tuán)呈橘黃色半透明,與散在的胰島細(xì)胞一起懸浮于培養(yǎng)基中(圖1A)。培養(yǎng)5 d后,胰島細(xì)胞呈類(lèi)圓形細(xì)胞,胞質(zhì)豐富,邊緣清晰,有的細(xì)胞團(tuán)邊緣有一紫色弧形反光帶,部分細(xì)胞團(tuán)已開(kāi)始鋪散開(kāi)貼壁,部分仍為細(xì)胞團(tuán),還有大量單個(gè)細(xì)胞貼壁或懸浮,可見(jiàn)成纖維細(xì)胞明顯較前減少,細(xì)胞生長(zhǎng)良好(圖1B)。
圖1 細(xì)胞接種第1天及第5天高倍鏡下表現(xiàn)Fig 1 The performance of high-powered microscope in the first and the fifth day of cell culture
表2 不同濃度鎢酸鈉實(shí)驗(yàn)組及對(duì)照組培養(yǎng)7天細(xì)胞計(jì)數(shù)結(jié)果Table 2 The cells count results of test and control groups treated by different concentration sodium tungstate(±s,×104,n=4)
表2 不同濃度鎢酸鈉實(shí)驗(yàn)組及對(duì)照組培養(yǎng)7天細(xì)胞計(jì)數(shù)結(jié)果Table 2 The cells count results of test and control groups treated by different concentration sodium tungstate(±s,×104,n=4)
*P <0.01 compared with control group.
group(μmol/L)day 1 day 3 day 5 day 7 control 2.00±0.00 3.00±0.33 6.00±1.03 7.33±0.65 50 2.00±0.00 3.63±0.42* 8.00±0.81* 9.63±0.98*150 2.00±0.00 3.75±0.43* 10.38±1.05* 11.00±1.00*300 2.00±0.00 4.38±0.51* 11.50±0.88* 12.13±1.03*500 2.00±0.00 4.88±0.42* 6.88±0.34* 8.13±0.88*700 2.00±0.00 4.50±0.35* 6.00±0.41* 6.13±0.68*
表3 鎢酸鈉干預(yù)實(shí)驗(yàn)組與對(duì)照組吸光度值結(jié)果Table 3 The absorption results of test and control groups treated by sodium tungstate(±s,A value,n=8)
表3 鎢酸鈉干預(yù)實(shí)驗(yàn)組與對(duì)照組吸光度值結(jié)果Table 3 The absorption results of test and control groups treated by sodium tungstate(±s,A value,n=8)
*P <0.01 compared with control group.
group day 1 day 3 day 5 day 7 control 0.256±0.007 0.481±0.021 0.840±0.0520.895±0.043 test 0.254±0.112 0.594±0.074* 1.173±0.024* 1.205±0.062*
細(xì)胞在培養(yǎng)第3~5天細(xì)胞數(shù)增長(zhǎng)最快,隨后進(jìn)入平臺(tái)期。濃度在50~500 μmol/L的鎢酸鈉處理組細(xì)胞數(shù)增加較明顯,而濃度增高至700 μmol/L時(shí)胰島細(xì)胞增長(zhǎng)反而受抑制(表2)。
隨著培養(yǎng)時(shí)間的增加,以第3~5天時(shí)實(shí)驗(yàn)組細(xì)胞增殖最明顯(P<0.01)(表3)。
在高糖或低糖刺激時(shí),鎢酸鈉干預(yù)組胰島素分泌水平及胰島素刺激指數(shù)(ISI)均較對(duì)照組顯著增高(P<0.05)(表4),提示鎢酸鈉增強(qiáng)新生豬胰島細(xì)胞功能,促進(jìn)胰島素分泌。
表4 實(shí)驗(yàn)組與對(duì)照組葡萄糖刺激實(shí)驗(yàn)結(jié)果Table 4 The glucose stimulation test results of test and control group(μU/mL)
鎢酸鈉處理3 d后,PCNA蛋白及Insulin蛋白表達(dá)分別為2.24±0.19和1.62±0.17,顯著高于對(duì)照組的1.17±0.13和0.37±0.08(P<0.01)(圖2)。
圖2 鎢酸鈉干預(yù)對(duì)新生豬胰島細(xì)胞PCNA、insulin蛋白表達(dá)的影響Fig 2 The PCNA and insulin protein expression after neonatal porcine islets treated by sodium tungstate
鎢酸鈉干預(yù)組PDX-1mRNA和GLUT-2mRNA分別為0.34±0.05和1.06±0.10,顯著高于對(duì)照組的0.11±0.03和0.13±0.02(P<0.01)(圖3)。
圖3 經(jīng)鎢酸鈉300μmol/L干預(yù)后實(shí)驗(yàn)組PDX-1、GLUT-2基因的mRNA表達(dá)Fig 3 The PDX-1 and GLUT-2 mRNA expression after test and control groups were treated by 300 μmol/L sodium tungstate
鎢酸鈉的降糖作用分別在1型、2型糖尿病動(dòng)物實(shí)驗(yàn)中已得到證實(shí),無(wú)論短期或長(zhǎng)期用藥,還是撤藥后相當(dāng)長(zhǎng)一段時(shí)間均有顯著效果[1,3-4],此外還有降血脂的作用,且無(wú)明顯肝腎毒副反應(yīng)[4],因而被認(rèn)為可能是一種很有潛力的新型降糖藥物。其降糖機(jī)制除與釩酸鹽相似的類(lèi)胰島素作用外,還可能與其具有促胰島素分泌和胰島細(xì)胞再生作用有關(guān)[2,5-6]。
鎢酸鈉促進(jìn)糖尿病動(dòng)物模型的基礎(chǔ)及葡萄糖刺激后血清胰島素的水平升高[5],在體外也促進(jìn)胰島素分泌、細(xì)胞內(nèi)胰島素含量增多及對(duì)葡萄糖刺激的高敏性[7]且胰島素mRNA表達(dá)翻倍。但迄今為止所有實(shí)驗(yàn)?zāi)P突揪窒拊谑箢?lèi),本實(shí)驗(yàn)在新生豬胰島細(xì)胞中觀(guān)察到類(lèi)似作用。
新生豬胰島作為主要的異種胰島移植細(xì)胞,具有較高的增殖及分化成熟的潛能[8],除了分裂增殖外,還能從胰腺導(dǎo)管細(xì)胞分化而來(lái)。鎢酸鈉在一定濃度內(nèi)有促進(jìn)新生豬胰島細(xì)胞生長(zhǎng)的作用,高濃度抑制,這與鎢酸鹽、釩酸鹽、鉬酸鹽對(duì)BRIN-BD11細(xì)胞影響相似[7]。在糖尿病鼠胰島及生殖細(xì)胞上也有類(lèi)似結(jié)果[1,6,9]。PDX-1 的功能主要是調(diào)控胰腺的分化發(fā)育、促進(jìn)胰島細(xì)胞再生及胰島素基因的轉(zhuǎn)錄。在STZ糖尿病新生鼠胰島中,觀(guān)察到鎢酸鈉促進(jìn)胰島細(xì)胞分化成熟,且PDX-1的磷酸化形式比例明顯增高。本實(shí)驗(yàn)也觀(guān)察到鎢酸鈉上調(diào)新生豬胰島細(xì)胞PDX-1mRNA表達(dá),提示除了自身分裂增殖外,上調(diào)PDX-1mRNA表達(dá)或促進(jìn)PDX-1磷酸化增強(qiáng)導(dǎo)管細(xì)胞分化再生也可能是鎢酸鈉促進(jìn)β細(xì)胞增殖的機(jī)制之一,可能通過(guò)P38活化促進(jìn)PDX-1磷酸化[10],也可能通過(guò)直接增強(qiáng)P42/P44磷酸化、激活MAPK通路促進(jìn)β細(xì)胞增殖[2]。
鎢酸鈉可改善胰島細(xì)胞功能及活性,但其作用機(jī)制尚不明確。本實(shí)驗(yàn)首次觀(guān)察到鎢酸鈉上調(diào)β細(xì)胞GLUT-2mRNA的表達(dá),也能通過(guò)ERK1/2途徑促進(jìn)GLUT-4的合成和作用[11],D加速葡萄糖轉(zhuǎn)運(yùn),鎢酸鈉可激活細(xì)胞內(nèi)腺苷酸環(huán)化酶及磷酸肌醇的活性,也可能通過(guò)關(guān)閉K+-ATP通道發(fā)揮促胰島素分泌作用[12],降低腸道上皮黏膜蔗糖酶及SGLT1的活性,減少高血糖對(duì)胰島細(xì)胞、腦組織氧化應(yīng)激損傷也可能是其降糖機(jī)制[4,13-14]。
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Sodium tungstate promotes the proliferation,differentiation and secretion of neonatal porcine islet cells in vitro
LIU Shu-yuan#,CHEN Ke,LIU Min,HE Hong-hui,XIE Yan-hong,XIE Xiao-yun,MO Zhao-hui*
(Dept.of Endocrinology,the Third Xiangya Hospital,Central South University,Changsha 410013,China)
ObjectiveTo investigate the possible mechanism of sodium tungstate in diabetic therapeutical effects and the protective function in neonatal porcine islet cellsin vitro,the effects of sodium tungstate on proliferation and differentiation of neonatal porcine islet cells was observedin vitroin our experiment.MethodsNeonatal porcine islets(NPIS)were cultured with various concentrations of sodium tungstate and carry out cell counting and MTT assay every other day.NPIS was exposed to the most proper concentration 300 μmol/L sodium tungstate for three days,and the glucose-stimulated insulin secrtion(GSIS)were detected,the expression of proliferating cell nuclear antigen(PCNA),insulin content in islet cells andPDX-1mRNA,GLUT-2mRNA were assayed by Western blot and RT-PCR respectively.ResultsThe group of islet cells were cultured with the dose of 300 μmol/L had the higher light absorption in MTT assay(P<0.05)and GSIS was significantly increased in the group.The protein expressions of PCNA and INSULIN content in the experimental group were 2.24±0.19 and 1.62±0.17 significantly higher than that in control group which were 1.17±0.13 and 0.37±0.08(P<0.05,P<0.01).The mRNA expressions ofPDX-1andGLUT-2were 0.34±0.05 and 1.06±0.10 in the experimental group significantly higher than that in control group which were 0.11±0.03 and 0.13±0.02(P<0.01).Conclusions 300μmol/L sodium tungstate has the advanced effect on promoting the proliferation and differentiation of neonatal porcine islet cell.It also has the effect in enhancing the function of insulin secretion.
sodium tungstate;neonatal porcine islet cell;proliferation and differentiation;the function of islet cell
R 335+.6
A
1001-6325(2012)09-1076-06
2010-11-01
2012-01-04
湖南省自然科學(xué)基金(06JJ5035)
*通信作者(corresponding author):MZH1964@126.COM#現(xiàn)工作單位:廈門(mén)大學(xué)附屬中山醫(yī)院內(nèi)分泌科