趙歡，周斌,2,3

優(yōu)博專欄

胰島beta細(xì)胞再生研究進(jìn)展

趙歡1，周斌1,2,3

1. 中國科學(xué)院分子細(xì)胞科學(xué)卓越創(chuàng)新中心/生物化學(xué)與細(xì)胞生物學(xué)研究所，上海 200031 2. 上?？萍即髮W(xué)生命科學(xué)與技術(shù)學(xué)院，上海 201210 3. 國科大杭州高等研究院生命與健康科學(xué)學(xué)院，杭州 310024

胰島beta細(xì)胞分泌胰島素調(diào)控體內(nèi)血糖水平，胰島beta細(xì)胞數(shù)量減少會導(dǎo)致糖尿病的發(fā)生。胰島移植是目前治療糖尿病的有效方法，但是目前仍然面臨供體短缺等巨大障礙，因此研究胰島beta細(xì)胞再生對于糖尿病的臨床治療具有深遠(yuǎn)意義。beta細(xì)胞的再生來源主要包括內(nèi)源性beta細(xì)胞增殖、多能干細(xì)胞分化和其他非beta細(xì)胞的轉(zhuǎn)分化。成體是否存在內(nèi)源性胰腺干細(xì)胞依然是領(lǐng)域內(nèi)亟待解決的重要科學(xué)問題之一。本文總結(jié)了與胰島beta細(xì)胞再生相關(guān)的研究發(fā)現(xiàn)與進(jìn)展，并討論了內(nèi)源性胰島beta細(xì)胞增殖、誘導(dǎo)多能干細(xì)胞分化、非胰島beta細(xì)胞重編程等方法在糖尿病治療中需要注意的問題和潛在應(yīng)用前景。

胰腺beta細(xì)胞；再生；分化；增殖

全球大約有5.37億人飽受糖尿病及其并發(fā)癥的困擾，糖尿病是一種以高血糖為主要病癥的代謝性疾病，主要包括1型和2型糖尿病。1型糖尿病有家族性發(fā)病的特點(diǎn)，主要由自身免疫系統(tǒng)缺陷引起的胰島beta細(xì)胞選擇性破壞和功能受損，胰島素分泌缺乏，患者發(fā)病年齡小，需要終生補(bǔ)充外源性胰島素[1,2]。2型糖尿病患者占總患者人數(shù)90%以上，發(fā)病年齡較晚，病癥相對較輕，多由于肥胖及飲食過多等不良生活習(xí)慣等因素引起，但也有研究發(fā)現(xiàn)2型糖尿病患者的胰島beta細(xì)胞數(shù)量顯著減少[1,2]。糖尿病可以引發(fā)一系列包括失明、腎衰竭、腦中風(fēng)及冠狀血管疾病等并發(fā)癥，給社會造成巨大醫(yī)療負(fù)擔(dān)?，F(xiàn)有的治療方法例如胰島細(xì)胞移植雖然有效，但具有供體短缺、免疫排斥反應(yīng)等缺點(diǎn)。因此，研究如何促進(jìn)beta細(xì)胞再生將有助于尋找治療糖尿病的新策略，是領(lǐng)域內(nèi)亟待解決的重要科學(xué)問題之一[1,2]。本文主要綜述了哺乳動物成體胰島beta細(xì)胞來源和產(chǎn)生新胰島beta細(xì)胞策略及其調(diào)控機(jī)制最新研究進(jìn)展，以期為相關(guān)人員提供參考。

1 成體胰島beta細(xì)胞來源

作為人體內(nèi)主要消化器官之一，胰腺具有調(diào)控機(jī)體能量消耗與代謝的重要作用，位于人體腹上區(qū)腹膜后間隙，形狀細(xì)長，連接十二指腸和脾臟，主要包括外分泌部和內(nèi)分泌胰島(又稱朗格漢斯島)，一層稀疏結(jié)締組織覆蓋在表面將胰腺分隔成許多小葉。胰腺外分泌部主要由腺泡細(xì)胞和導(dǎo)管細(xì)胞構(gòu)成，腺泡細(xì)胞呈泡狀，可以分泌多種消化酶沿胰腺導(dǎo)管進(jìn)入小腸，參與分解脂肪、蛋白質(zhì)和碳水化合物等[1]。胰島體積很小，成緊密球狀組織，零散分布于外分泌部中，主要包括alpha細(xì)胞、beta細(xì)胞、delta細(xì)胞和PP細(xì)胞。胰島beta細(xì)胞可以分泌胰島素，與alpha細(xì)胞分泌的胰高血糖素通過胰島內(nèi)血管網(wǎng)絡(luò)直接進(jìn)入血液循環(huán)，協(xié)同維持機(jī)體血糖穩(wěn)定[1]。目前，對于成體胰島beta細(xì)胞來源主要有兩種觀點(diǎn)：原有beta細(xì)胞的自我復(fù)制和非beta細(xì)胞的轉(zhuǎn)分化(包括干細(xì)胞的分化)。

1.1 主要來源

細(xì)胞增殖、分化和轉(zhuǎn)分化等在機(jī)體生長發(fā)育及病理狀況下發(fā)揮著重要作用，準(zhǔn)確解析體內(nèi)細(xì)胞命運(yùn)有助于人們深入了解這些生物學(xué)過程[3]。成體胰島beta細(xì)胞來源的研究歷史悠久，研究對象主要集中于嚙齒類動物例如小鼠()。早期研究依賴于H3胸苷嘧啶摻入實(shí)驗(yàn)，提出成體胰島內(nèi)分泌細(xì)胞屬于可以通過自我復(fù)制來維持的分化細(xì)胞[4,5]，然而2002年的一項(xiàng)研究工作通過組織形態(tài)觀察發(fā)現(xiàn)胰島細(xì)胞存在干細(xì)胞來源[6]，此外也有研究發(fā)現(xiàn)胰島干細(xì)胞可能存在于骨髓[7]、胰島內(nèi)[8]及胰腺導(dǎo)管上皮細(xì)胞[9]。除了自我復(fù)制和干細(xì)胞分化，還有一種觀點(diǎn)認(rèn)為beta細(xì)胞是來自于胰腺腺泡細(xì)胞[10]和胰島內(nèi)其他非beta細(xì)胞的內(nèi)分泌細(xì)胞[11]的轉(zhuǎn)分化。然而，以上這些對于胰島內(nèi)分泌細(xì)胞來源的研究與結(jié)論都是基于靜態(tài)組織學(xué)層面的觀察分析，而不是對體內(nèi)細(xì)胞譜系的直接分析。

隨著遺傳譜系示蹤技術(shù)的出現(xiàn)和發(fā)展，該技術(shù)最初用于經(jīng)典的發(fā)育生物學(xué)領(lǐng)域，后來越來越多地用于干細(xì)胞科學(xué)研究，該技術(shù)可以探究體內(nèi)細(xì)胞遷移、增殖和命運(yùn)轉(zhuǎn)變等過程[12]。主要原理是基于位點(diǎn)特異性重組酶包括Cre/loxP等，當(dāng)以組織特異性方式激活Cre重組酶，就可以使特定細(xì)胞表達(dá)報(bào)告基因，這種標(biāo)記是可遺傳且永久性的，即使該細(xì)胞的子代細(xì)胞發(fā)生轉(zhuǎn)分化不再表達(dá)Cre，細(xì)胞譜系類型發(fā)生變化，其子代細(xì)胞也會持續(xù)表達(dá)報(bào)告基因，因此是解析體內(nèi)細(xì)胞命運(yùn)可塑性的金標(biāo)準(zhǔn)工具[12]。當(dāng)把Cre同源重組酶和雌激素受體結(jié)合就可以構(gòu)成誘導(dǎo)型CreER，只有在他莫昔芬(Tamoxifen)誘導(dǎo)的條件下，Cre同源重組酶才能入核發(fā)揮作用，可以實(shí)現(xiàn)定時控制其表達(dá)以研究特定細(xì)胞在體內(nèi)的起源與命運(yùn)[12]。

2004年，一項(xiàng)里程碑式的研究工作被報(bào)道。Dor等[13]利用譜系示蹤技術(shù)，發(fā)現(xiàn)小鼠成體胰島beta細(xì)胞主要來源于自我復(fù)制而非干細(xì)胞分化。在這項(xiàng)工作中，研究人員構(gòu)建了beta細(xì)胞特異性驅(qū)動小鼠，在小鼠成體后給予他莫昔芬注射，特異性誘導(dǎo)胰島beta細(xì)胞標(biāo)記人堿性磷酸酶蛋白，因?yàn)檫@種遺傳重組是發(fā)生在DNA水平，這些已有的beta細(xì)胞及它們所有的子代細(xì)胞都會被永久標(biāo)記，在經(jīng)過一段時間后收取小鼠組織，通過組織化學(xué)染色檢測此時胰島beta細(xì)胞標(biāo)記蛋白的表達(dá)情況。理論上，如果是新的beta細(xì)胞來源于干細(xì)胞或者祖細(xì)胞，這些新的beta細(xì)胞就不會表達(dá)標(biāo)記蛋白，那么beta細(xì)胞的標(biāo)記比例會降低；如果新的beta細(xì)胞來源于原有beta細(xì)胞的自我復(fù)制，胰島內(nèi)標(biāo)記的beta細(xì)胞比例就會保持穩(wěn)定。研究人員收取了誘導(dǎo)標(biāo)記后2.5、4、6、9和12個月的小鼠胰腺組織，發(fā)現(xiàn)beta細(xì)胞標(biāo)記效率在這些時間點(diǎn)維持穩(wěn)定，說明成體胰島beta細(xì)胞來自于原有beta細(xì)胞的自我復(fù)制[13]。

2007年，另一項(xiàng)里程碑式研究工作通過借助獨(dú)立于譜系示蹤技術(shù)的新型雙胸腺嘧啶類似物標(biāo)記策略，證明小鼠出生后胰島beta細(xì)胞的產(chǎn)生來自于自我更新而不是祖細(xì)胞分化[14]。研究人員在不同時間往小鼠飲用水中添加兩種不同的胸腺嘧啶類似物，理論上，每種類似物的摻入都代表一次細(xì)胞分裂，如果細(xì)胞主要來源于特化的干/祖細(xì)胞，那么最近分裂的細(xì)胞就會經(jīng)歷多次細(xì)胞分裂而被標(biāo)記上兩種類似物；相反，如果細(xì)胞主要通過自我更新來維持，那么最近分裂的細(xì)胞就不會經(jīng)歷多次細(xì)胞分裂，只能摻入一種類似物。借助該技術(shù)，研究人員發(fā)現(xiàn)胃腸上皮細(xì)胞和皮膚可以檢測到大量有兩種類似物摻入的細(xì)胞，而胰島beta細(xì)胞只有一種標(biāo)記，因此認(rèn)為beta細(xì)胞是來源于自我更新[14]。

此后，國內(nèi)外有多個研究組利用不同的遺傳工具支持這一觀點(diǎn)[14～20]。這些研究工作一致認(rèn)為，哺乳動物胚胎期存在胰島干細(xì)胞分化及其他細(xì)胞去分化來源的beta細(xì)胞新生，而這一現(xiàn)象在出生后就急劇減少，成體胰島beta細(xì)胞的自我復(fù)制是其產(chǎn)生的主要途徑(圖1)。

以上工作主要是研究胰腺生理穩(wěn)態(tài)及損傷模型，包括胰腺部分切除、胰腺導(dǎo)管結(jié)扎和化學(xué)藥物引起beta細(xì)胞損傷等(圖1)。研究發(fā)現(xiàn)，哺乳動物在妊娠期間或肥胖條件下可以觀察到胰島增生，beta細(xì)胞數(shù)量顯著增加[21,22]。在胰腺切除術(shù)中，切除90%的胰腺不會影響大鼠()的葡萄糖穩(wěn)態(tài)[23,24]，說明嚙齒類動物胰腺有很強(qiáng)的儲存與再生能力，而人類中切除50%左右的胰腺就會引起胰島素依賴型糖尿病[25]。胰腺導(dǎo)管結(jié)扎可以引起外分泌部腺泡細(xì)胞大量死亡，引起類似胰腺炎癥狀，但普遍認(rèn)為該模型不會對內(nèi)分泌胰島造成嚴(yán)重傷害和引起再生[26]?；瘜W(xué)藥物例如鏈脲佐菌素(streptozotocin, STZ)等誘導(dǎo)部分beta細(xì)胞損傷和消融，其原理是STZ作為葡萄糖類似物可以通過葡萄糖轉(zhuǎn)運(yùn)蛋白2 (glucose transporter type 2, GLUT2)發(fā)揮作用使DNA烷基化，腺苷三磷酸(adenosine tri-phosphate, ATP)產(chǎn)生減少，小鼠胰島素缺乏，表現(xiàn)出1型糖尿病癥狀[27,28]。

圖1 胰島beta細(xì)胞的自我復(fù)制

動物研究表明，beta細(xì)胞復(fù)制是穩(wěn)態(tài)、妊娠、損傷和胰島素抵抗中再生和修復(fù)的主要模式。

1.2 成體胰島beta細(xì)胞干細(xì)胞爭議

胰腺干細(xì)胞指的是可以分化產(chǎn)生多個胰腺細(xì)胞譜系，同時具有自我更新能力的細(xì)胞。在哺乳動物胚胎發(fā)育過程中，胰腺干細(xì)胞存在于胰腺導(dǎo)管上皮，可以分化產(chǎn)生胰腺內(nèi)分泌部和外分泌部[29]。一個尚未完全回答的重要科學(xué)問題是成體動物包括人類體內(nèi)是否存在內(nèi)源性胰腺干細(xì)胞，可以分化產(chǎn)生胰島beta細(xì)胞？因?yàn)閎eta細(xì)胞的大量丟失會造成人體內(nèi)分泌失調(diào)及不可逆的糖尿病，尋找和鑒定胰島beta細(xì)胞干細(xì)胞可以為糖尿病臨床治療提供理論依據(jù)和新的研究方向，具有深遠(yuǎn)意義。

一些譜系示蹤研究工作支持成體胰島beta細(xì)胞干細(xì)胞存在。例如，在胰管結(jié)扎(pancreatic duct ligation, PDL)損傷后，成年導(dǎo)管基因神經(jīng)元素3 (neurogenin3, Ngn3)陽性細(xì)胞可重新激活，增殖并分化為beta細(xì)胞[30]。成年胰管碳酸酐酶II (carbonic anhydrase II)陽性細(xì)胞在胰管結(jié)扎模型損傷后也可作為祖細(xì)胞生成beta細(xì)胞[31]。同樣，少量成體胰腺特異轉(zhuǎn)錄因子1a (pancreas specific transcription factor 1a, Ptf1a)陽性腺泡細(xì)胞在PDL后可快速重編程生成導(dǎo)管細(xì)胞，并可以分化產(chǎn)生少量胰島素陽性beta細(xì)胞[32]。此外，其他研究也提示存在成體胰腺干細(xì)胞或祖細(xì)胞，包括CD133 (prominin 1)陽性細(xì)胞[33]、醛脫氫酶1A1 (aldehyde dehydrogenase 1a1, Aldh1a1)陽性細(xì)胞[34]、胰腺-十二指腸同源盒基因-1 (pancreatic and duodenal homeobox-1, Pdx1)陽性細(xì)胞[35]、SRY (性別決定區(qū)Y)框9 (SRY (sex determining region Y)-box 9, Sox9)陽性導(dǎo)管細(xì)胞[36]、蛋白C受體(protein C receptor, Procr)陽性細(xì)胞[37]及Ngn3陽性生長激素抑制素(somatostatin, Sst)陽性導(dǎo)管細(xì)胞[38]。然而，一些研究組基于胰腺導(dǎo)管細(xì)胞或腺泡細(xì)胞特異標(biāo)記以及廣譜性細(xì)胞標(biāo)記介導(dǎo)的遺傳譜系示蹤研究結(jié)果并不支持成體胰島干細(xì)胞的存在[13～20]。

存在爭議的可能原因是所謂的干細(xì)胞啟動子在體內(nèi)標(biāo)記或許并不特異，基于Cre/loxP同源重組系統(tǒng)的遺傳譜系示蹤技術(shù)的準(zhǔn)確性取決于Cre小鼠的特異性，因此進(jìn)行遺傳譜系示蹤研究時需要非常嚴(yán)格的檢驗(yàn)[39]。例如，當(dāng)使用A-Cre(ER)小鼠來標(biāo)記細(xì)胞B，最終觀察到“變成”了細(xì)胞C，如果要得出細(xì)胞B可以產(chǎn)生細(xì)胞C的結(jié)論，需要先驗(yàn)證在標(biāo)記最初A-Cre(ER)小鼠對細(xì)胞B標(biāo)記的特異性，排除A啟動子在細(xì)胞C中的“泄露”表達(dá)以及實(shí)驗(yàn)條件是否會誘導(dǎo)A啟動子在細(xì)胞C中的啟動，從而導(dǎo)致A-Cre(ER)小鼠對細(xì)胞C的預(yù)先標(biāo)記，影響正確結(jié)論的得出。所以，提出胰腺干細(xì)胞貢獻(xiàn)產(chǎn)生beta細(xì)胞結(jié)論的前提是該干細(xì)胞的啟動子不會預(yù)先標(biāo)記beta細(xì)胞[39～41]。因此，是否存在成體胰島beta細(xì)胞干細(xì)胞仍有待進(jìn)一步探究。

2 胰島beta細(xì)胞再生策略

2.1 內(nèi)源性胰島beta細(xì)胞增殖

嚙齒類動物的胰島beta細(xì)胞具有較高增殖率。研究發(fā)現(xiàn)，1月齡大鼠胰島beta細(xì)胞大約每天有4%增殖率，而到了7月齡增殖率就降到了大約每天0.5%[42,43]。妊娠和肥胖可以促進(jìn)小鼠胰島beta細(xì)胞增殖，可能是由于胰島素和葡萄糖升高直接刺激beta細(xì)胞增殖，涉及多條信號通路[44～47]。但是，通過增殖標(biāo)記物Ki67和增殖細(xì)胞核抗原(prolife-rating cell nuclear antigen, PCNA)組織染色，在健康、懷孕及肥胖的人類成體胰腺中很少發(fā)現(xiàn)有增殖的beta細(xì)胞[25,48,49]。有研究發(fā)現(xiàn)小鼠胰島beta細(xì)胞具有異質(zhì)性，表達(dá)Flattop的beta細(xì)胞具有成熟beta細(xì)胞特征，而不表達(dá)Flattop的beta細(xì)胞具有更強(qiáng)的細(xì)胞增殖能力。單細(xì)胞測序技術(shù)的發(fā)展與應(yīng)用擴(kuò)展了人們對beta細(xì)胞增殖和異質(zhì)性的理解，很多研究證明beta細(xì)胞具有異質(zhì)性[51]。研究人員發(fā)現(xiàn)人體內(nèi)胰島beta細(xì)胞具有基因表達(dá)和功能不同的4種亞型[52]。目前臨床上對于beta細(xì)胞再生療法的需求巨大，促進(jìn)內(nèi)源性beta細(xì)胞增殖依然是目前補(bǔ)充beta細(xì)胞的一種直接又簡單的解決方案。

目前，該方面的研究主要集中于動物模型，大量研究工作發(fā)現(xiàn)，多種生長因子和促有絲分裂劑包括表皮生長因子、肝細(xì)胞生長因子、胰高血糖素樣肽、胰島素樣生長因子等可以促進(jìn)beta細(xì)胞增殖[53～61]，這些因子在人體內(nèi)是否可以促進(jìn)beta細(xì)胞增殖還有待進(jìn)一步驗(yàn)證。高通量化合物篩選發(fā)現(xiàn)雙特異性酪氨酸磷酸化可以刺激調(diào)節(jié)激酶1A (DYRK1A)抑制劑，不僅可以在小鼠體內(nèi)有效促進(jìn)beta細(xì)胞增殖，還可以促進(jìn)體外培養(yǎng)的人beta細(xì)胞和體內(nèi)移植人beta細(xì)胞增殖[62～64]。近期發(fā)表在雜志的一項(xiàng)重磅研究工作發(fā)現(xiàn)在beta細(xì)胞中敲除胰島素抑制受體(insulin inhibitory receptor, inceptor)和利用單克隆抗體阻斷該受體發(fā)揮作用，可以增加功能性beta細(xì)胞的數(shù)量[65]。

人類成體胰島beta細(xì)胞具有進(jìn)入細(xì)胞周期所必需的分子元件，比如細(xì)胞周期蛋白、細(xì)胞周期蛋白依賴性激酶等，直接操縱這些分子機(jī)制可以迫使人類beta細(xì)胞增殖，但是細(xì)胞周期基因突變會導(dǎo)致胰腺內(nèi)分泌增生，甚至引起胰島細(xì)胞瘤[66～68]。因此，如何安全有效地促進(jìn)人類胰島beta細(xì)胞增殖并正常發(fā)揮功能，同時要確保不會引起腫瘤形成等問題，依然是領(lǐng)域內(nèi)的一個重要研究方向。

2.2 誘導(dǎo)多能干細(xì)胞分化

胰島移植是近年來有效治療糖尿病的方法之一，但是面臨著供體不足等問題，限制了該方法的普及使用。因此，從人類多能干細(xì)胞中誘導(dǎo)分化提取胰島細(xì)胞移植是一種很有應(yīng)用前景的治療糖尿病的方法。2006年日本學(xué)者首次報(bào)道了利用逆轉(zhuǎn)錄病毒將4個轉(zhuǎn)錄因子(Oct4、Sox2、c-Myc和Klf4)導(dǎo)入成體成纖維細(xì)胞誘導(dǎo)形成多能干細(xì)胞的研究[69]，開創(chuàng)了疾病治療領(lǐng)域一個新的重要方向。通過轉(zhuǎn)錄因子的組合，研究人員實(shí)現(xiàn)了誘導(dǎo)人類胚胎干細(xì)胞向胰島細(xì)胞的分化[70]，展現(xiàn)出干細(xì)胞在糖尿病治療中的應(yīng)用潛力。但是在這項(xiàng)研究工作中，分化產(chǎn)生的是一組表達(dá)混合激素的細(xì)胞，而不是真正意義的成熟beta細(xì)胞。后續(xù)，研究人員誘導(dǎo)生產(chǎn)出了可以對葡萄糖刺激有反應(yīng)的內(nèi)分泌胰島，當(dāng)把人類胚胎干細(xì)胞來源的PDX1+祖細(xì)胞移植入小鼠體內(nèi)時，可以在體內(nèi)分化形成功能性beta細(xì)胞，逆轉(zhuǎn)糖尿病[71]。

長期以來，通過對誘導(dǎo)因子和化合物的不斷優(yōu)化，并且借助于三維培養(yǎng)模式，研究人員設(shè)計(jì)了更加復(fù)雜的分化方案，使產(chǎn)生的細(xì)胞簇具有更加顯著的胰島形態(tài)和功能相似性[72～81]。然而，到目前為止，由干細(xì)胞誘導(dǎo)產(chǎn)生的beta細(xì)胞還不具有成熟功能，對胰島素分泌調(diào)節(jié)能力較差。Balboa等[82]進(jìn)一步優(yōu)化分化方案，與常用分化方案不同之處主要包括：在粘附狀態(tài)下分化人多能干細(xì)胞到胰腺祖細(xì)胞期；優(yōu)化第四階段(后前腸到胰腺祖細(xì)胞階段)和微孔聚集步驟；改進(jìn)第七階段(最終成熟階段)在懸浮培養(yǎng)條件下進(jìn)行。通過代謝功能研究和單細(xì)胞轉(zhuǎn)錄組學(xué)，首次證實(shí)可以誘導(dǎo)形成在結(jié)構(gòu)和功能上都非常成熟的胰島，并且對其功能特性進(jìn)行了非常詳細(xì)地探究與表述，也證實(shí)移植到小鼠體內(nèi)的誘導(dǎo)beta細(xì)胞可以有效調(diào)控體內(nèi)葡萄糖代謝，這一成果使該技術(shù)更容易應(yīng)用于疾病的治療[82]。但是，干細(xì)胞誘導(dǎo)分化產(chǎn)生beta細(xì)胞技術(shù)到臨床應(yīng)用還依然存在一些重要挑戰(zhàn)，例如，如何進(jìn)一步完善大規(guī)模制造和進(jìn)行質(zhì)量控制，如何防止移植后的免疫排斥等問題，需要進(jìn)一步探究。

2.3 非胰島beta細(xì)胞重編程

通過患者本身終末分化的非胰島beta細(xì)胞重編程產(chǎn)生beta細(xì)胞，可以有效規(guī)避體內(nèi)免疫排斥反應(yīng)等問題，具有重要意義，也是糖尿病臨床治療的有效途徑之一。Thorel等[83]和Chera等[84]相繼發(fā)現(xiàn)，當(dāng)利用白喉毒素清除小鼠體內(nèi)99%的beta細(xì)胞，幾個月后可以觀察到beta細(xì)胞的緩慢恢復(fù)，新的beta細(xì)胞來源于胰島alpha或delta細(xì)胞的轉(zhuǎn)分化，這種轉(zhuǎn)分化與小鼠年齡有很大的相關(guān)性。表觀遺傳因子Arx和Pax4在這種轉(zhuǎn)分化過程中發(fā)揮了關(guān)鍵作用[85,86]。后續(xù)研究發(fā)現(xiàn)，長期用gamma氨基丁酸(GABA)處理也可以促進(jìn)小鼠alpha細(xì)胞向beta細(xì)胞轉(zhuǎn)變[87]，并且位于胰島外圍的一個特殊細(xì)胞群可能是alpha細(xì)胞向beta細(xì)胞轉(zhuǎn)變的一個中間態(tài)[88,89]。Alpha細(xì)胞和beta細(xì)胞都位于胰島內(nèi)部，有類似的形態(tài)結(jié)構(gòu)，而且在胚胎發(fā)育過程中來源于相同的前體祖細(xì)胞[90]，或許是重編程形成beta細(xì)胞的理想來源，但是目前還沒有確切的研究結(jié)果證明人體內(nèi)是否存在這種胰島內(nèi)細(xì)胞類型間的轉(zhuǎn)化。

對胰腺發(fā)育進(jìn)程各階段相關(guān)轉(zhuǎn)錄因子的研究可以有助于對終末分化細(xì)胞重編程產(chǎn)生beta細(xì)胞的探索，具有重要意義。發(fā)育過程中涉及很多轉(zhuǎn)錄因子，例如：胰腺-十二指腸同源盒基因-1 (pancreatic and duodenal homeobox-1, Pdx1)是胰腺發(fā)育、beta細(xì)胞增殖、成熟和發(fā)揮功能所必需的重要轉(zhuǎn)錄因子，胚胎時期表達(dá)于早期胰腺芽體上皮細(xì)胞，具有多能分化潛能[91]；肌腱膜纖維肉瘤癌基因家族蛋白A (v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A, MafA)是可以與INS結(jié)合，是調(diào)節(jié)beta細(xì)胞代謝的一個轉(zhuǎn)錄因子，相對特異地表達(dá)于胰島beta細(xì)胞[92～94]；Ngn3是胚胎發(fā)育過程中胰腺內(nèi)分泌祖細(xì)胞表達(dá)的關(guān)鍵轉(zhuǎn)錄因子之一，參與調(diào)控啟動胰腺前體細(xì)胞向內(nèi)分泌細(xì)胞分化過程[95～97]。

由于胰腺腺泡細(xì)胞數(shù)量眾多，普遍認(rèn)為是體內(nèi)轉(zhuǎn)分化形成beta細(xì)胞的理想來源之一。2008年，Zhou等[98]借助腺病毒載體將3種關(guān)鍵的胰島beta細(xì)胞轉(zhuǎn)錄因子Ngn3、Pdx1和MafA組合表達(dá)，成功將小鼠外分泌部腺泡細(xì)胞重編程為beta細(xì)胞樣細(xì)胞，這一結(jié)論在體外也得到證實(shí)[99]，誘導(dǎo)形成的beta細(xì)胞樣細(xì)胞不僅表達(dá)成熟beta細(xì)胞標(biāo)志基因，還具有逆轉(zhuǎn)糖尿病的能力，為糖尿病治療開辟了新的研究方向[98～100]。肝和胰島在早期胚胎發(fā)育過程具有相同來源，研究人員發(fā)現(xiàn)Ngn3、Pdx1和MafA三個轉(zhuǎn)錄因子共同表達(dá)可以誘導(dǎo)Sox9+肝細(xì)胞表達(dá)胰島素[101]，轉(zhuǎn)錄因子Tgif2可以促進(jìn)肝細(xì)胞重編程進(jìn)入胰腺祖細(xì)胞狀態(tài)[102]。

2018年，Xiao等[103]借助腺相關(guān)病毒向胰島導(dǎo)入Pdx1和MafA，基于遺傳譜系示蹤技術(shù)證明可以成功將胰島中的alpha細(xì)胞重編程形成可以分泌胰島素的beta細(xì)胞[103]。小鼠體細(xì)胞重編程產(chǎn)生beta細(xì)胞的其他研究還包括胰腺導(dǎo)管細(xì)胞[104]、胃腸道上皮細(xì)胞[105]、NGN3+腸道內(nèi)分泌祖細(xì)胞[106]、腸腺窩細(xì)胞[107]等。此外，還有一些研究報(bào)道了在體外對人類體細(xì)胞(包括胰腺外分泌細(xì)胞、成纖維細(xì)胞和脂肪細(xì)胞等)重編程產(chǎn)生可以分泌胰島素的beta細(xì)胞[108～114]。

目前，體細(xì)胞重編程產(chǎn)生beta細(xì)胞應(yīng)用于臨床的主要挑戰(zhàn)之一在于如何安全可靠地高效生產(chǎn)具有成熟功能的beta細(xì)胞，并且需要檢測在移植后是否可以長期保持穩(wěn)定的功能性和表觀遺傳狀態(tài)，因此依然需要不斷優(yōu)化重編程方案，同時解決病毒轉(zhuǎn)染的安全問題。

3 結(jié)語與展望

探討胰島beta細(xì)胞再生的來源和機(jī)制對于糖尿病等的治療具有重要意義。但值得注意的是，應(yīng)謹(jǐn)慎將在嚙齒動物模型得到的結(jié)論推廣應(yīng)用于臨床治療，因?yàn)槿祟惡蛧X類動物的胰島在結(jié)構(gòu)和基因表達(dá)上存在一些差異。例如，小鼠胰島beta細(xì)胞主要集中在胰島核心，但是在人類胰島中beta細(xì)胞分布更加均勻；人類胰島含有更高比例的alpha細(xì)胞和更低比例的beta細(xì)胞；人類胰島使用GLUT1而不是GLUT2作為主要的葡萄糖轉(zhuǎn)運(yùn)體[115,116]等。盡管目前對于促進(jìn)胰島beta細(xì)胞再生研究方面取得了巨大進(jìn)展，但依然存在一些關(guān)鍵問題，是以后研究工作的重點(diǎn)。例如：成年動物包括人類胰腺中究竟是否存在可以分化產(chǎn)生beta細(xì)胞的干細(xì)胞？胰島beta細(xì)胞在體內(nèi)是否具有功能異質(zhì)性？重編程產(chǎn)生beta細(xì)胞的安全性如何保證？如何獲得大規(guī)模的高質(zhì)量成熟beta細(xì)胞？除了這些方法的安全有效性以外，這些分化或重編程beta細(xì)胞用于臨床研究的另一個關(guān)鍵因素是在糖尿病患者特別是1型糖尿病患者自身免疫缺陷環(huán)境中是否可以正常發(fā)揮細(xì)胞功能。因此，對于beta細(xì)胞再生的基礎(chǔ)研究也應(yīng)該進(jìn)一步加強(qiáng)與免疫學(xué)領(lǐng)域的合作，如聯(lián)合使用免疫抑制劑，找到可以產(chǎn)生自然抵抗自身免疫的胰島的理想方法。

對于胰島beta細(xì)胞穩(wěn)態(tài)維持和如何促進(jìn)其再生及內(nèi)在調(diào)節(jié)信號通路的基礎(chǔ)研究將進(jìn)一步為糖尿病的臨床治療提供理論基礎(chǔ)和新的研究方向，對于上述重要科學(xué)問題，新的技術(shù)手段將在推進(jìn)這些研究中發(fā)揮重要作用。例如構(gòu)建人源化小鼠模型和人類胰島類器官作為人類胰腺的替代品，可以幫助在嚙齒類動物研究中獲得的促進(jìn)beta細(xì)胞再生的研究發(fā)現(xiàn)進(jìn)行驗(yàn)證；三維胰腺類器官培養(yǎng)有助于在體外大量形成成熟完善的胰島樣細(xì)胞；CRISPR/Cas9技術(shù)可以幫助進(jìn)行體內(nèi)基因文庫篩選和快速構(gòu)建模型進(jìn)行驗(yàn)證；單細(xì)胞測序技術(shù)可以為胰島beta細(xì)胞異質(zhì)性和分化/重編程產(chǎn)生的beta細(xì)胞的基因表達(dá)驗(yàn)證提供非常強(qiáng)大的技術(shù)支持等。

綜上所述，目前對胰島beta細(xì)胞再生的研究還有很大探索空間，相信隨著科學(xué)技術(shù)的進(jìn)步和發(fā)展，會為糖尿病臨床治療與治愈開辟新的研究道路，為患者帶來希望。

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Pancreatic beta cells regeneration

Huan Zhao1, Bin Zhou1,2,3

Insulin-secreting pancreatic beta cells are important for the regulation of glucose metabolism. Loss of beta cells could lead to diabetes. So far, islet transplantation is an effective way for diabetes therapy, but the shortage of donors limits its extensive application. Therefore, study on beta cell regeneration has great significance for the clinical treatment of diabetes. The major cellular sources of beta cells mainly include endogenous beta cell proliferation, pluripotent stem cell differentiation, and trans-differentiation from other non-beta cells. The existence of endogenous pancreatic stem cells or progenitors in adults remains controversial in the field. In this review, we briefly summarize the research advances of beta cell regeneration and discuss the potential problems of these approaches, to improve our understanding on the mechanisms of beta cell regeneration and provide theoretical information for clinical application.

pancreatic beta cell; regeneration; differentiation; proliferation

2022-03-16;

2022-04-05;

2022-04-21

國家自然科學(xué)基金項(xiàng)目(編號：31730112，91849202，9216800001)和賽諾菲優(yōu)秀青年人才獎勵基金資助[Supported by the National Natural Science Foundation of China (Nos. 31730112, 91849202, 9216800001) and the Sanofi Scholarship Program]

趙歡，博士，研究方向：遺傳譜系示蹤技術(shù)。E-mail: zhaohuan@sibs.ac.cn

周斌，研究員，博士生導(dǎo)師，研究方向：譜系示蹤與細(xì)胞命運(yùn)可塑性。E-mail: zhoubin@sibs.ac.cn

10.16288/j.yczz.22-072

趙歡，2015—2020年就讀于中國科學(xué)院分子細(xì)胞科學(xué)卓越創(chuàng)新中心，在周斌課題組攻讀博士學(xué)位，目前在中國科學(xué)院分子細(xì)胞科學(xué)卓越創(chuàng)新中心進(jìn)行博士后訓(xùn)練。博士期間，主要研究遺傳譜系示蹤技術(shù)的開發(fā)與應(yīng)用，主要工作有：利用譜系示蹤技術(shù)靶向腫瘤新生血管，進(jìn)一步揭示腫瘤惡性增殖與血管新生的關(guān)系；使用CRISPR/Cas9系統(tǒng)構(gòu)建家族性高膽固醇血癥小鼠模型并靶向致病基因，發(fā)現(xiàn)能夠改善動脈粥樣硬化等相關(guān)疾病表型；開發(fā)了能夠同時示蹤胰島beta細(xì)胞和非beta細(xì)胞的譜系示蹤新技術(shù)，為糖尿病臨床治療研究提供了重要的理論基礎(chǔ)和研究新思路。博士論文《遺傳譜系示蹤腫瘤血管新生和胰腺β細(xì)胞》獲得2021年中國科學(xué)院優(yōu)秀博士生論文。

(責(zé)任編委: 陳帥)