葉海峰 綜述,李小燕,李 薇,鄭月慧△ 審校
(1.南昌大學(xué)江西醫(yī)學(xué)院 330031;2.江西省生殖生理與病理重點實驗室,南昌 330031)
卵巢生殖干細(xì)胞的發(fā)現(xiàn)、成功分離及建系打破了壟斷生殖醫(yī)學(xué)界一百多年的卵泡池固定學(xué)說,并為有效地增加原始卵泡池、改善卵巢功能和延緩卵巢衰老帶來了前所未有的希望。傳統(tǒng)觀點認(rèn)為機(jī)體和組織器官的衰老是由于干細(xì)胞的衰老導(dǎo)致的,但在卵巢衰老中,卵巢干細(xì)胞巢的衰老和退化被認(rèn)為是導(dǎo)致卵巢生殖功能衰退的主要因素,而并非只是由于卵巢生殖干細(xì)胞本身的衰老所致。本文將論述哺乳動物卵巢生殖干細(xì)胞巢的衰老對生殖功能產(chǎn)生的影響。
干細(xì)胞巢就是干細(xì)胞微環(huán)境,目前哺乳動物造血干細(xì)胞巢、小腸干細(xì)胞巢、神經(jīng)元干細(xì)胞巢、精元干細(xì)胞巢的結(jié)構(gòu)和功能已經(jīng)研究的相對透徹[1-4],但哺乳動物卵巢干細(xì)胞巢的相關(guān)研究及其衰老對卵巢功能的影響還有待進(jìn)一步研究。
持續(xù)了半個多世紀(jì)的傳統(tǒng)觀點認(rèn)為,雌性哺乳動物卵泡池重大的卵母細(xì)胞在出生后便已經(jīng)固定,青春期后開始不斷減少直至全部耗竭[5-6]。然而在2004年,JOHNSON等[7]在測定小鼠不同天數(shù)閉鎖卵泡數(shù)和未閉鎖卵泡數(shù)時,結(jié)果發(fā)現(xiàn)卵泡閉鎖的發(fā)生率要高于未閉鎖卵泡數(shù)量的減少率,因此便推測卵巢中存在具有增殖能力的的細(xì)胞,這對壟斷生殖醫(yī)學(xué)界的卵泡池固定理論提出了嚴(yán)峻的挑戰(zhàn)。此后,各國的學(xué)者們紛紛利用不同的方法進(jìn)行反復(fù)驗證,雖然目前仍有少數(shù)學(xué)者對哺乳卵巢干細(xì)胞仍持懷疑態(tài)度[8-9],但研究者通過干細(xì)胞分離純化、體外培養(yǎng)、移植、轉(zhuǎn)基因和體內(nèi)世系追蹤等方法,在多種哺乳動物包括人類的卵巢均發(fā)現(xiàn)卵巢生殖干細(xì)胞的存在[10-12]。
卵巢生殖干細(xì)胞是來自于卵巢皮層的雙潛能干細(xì)胞,能通過對稱分裂產(chǎn)生一個新的干細(xì)胞或不對稱分裂分化為卵巢生殖細(xì)胞和原始顆粒細(xì)胞[13]。卵巢生殖干細(xì)胞在光鏡下呈圓形,約15~20 μm,數(shù)目稀少,經(jīng)傳代增殖后可見成串或成簇存在,經(jīng)PCR檢測表達(dá)果蠅vasa基因同源類似物(MVH)、把具體結(jié)合轉(zhuǎn)錄因子4(OCT4)、同源框蛋白質(zhì)(Mango)、干擾素誘導(dǎo)的跨膜蛋白(Fragilis)、二肽基肽(Stella)、酪氨酸激酶受體(C-kit)等多種生殖細(xì)胞和干細(xì)胞標(biāo)志物。
干細(xì)胞巢即干細(xì)胞周圍所生存的微環(huán)境,由SCHOFIELD[14]在研究造血干細(xì)胞的特殊環(huán)境時提出的,后來在消化系統(tǒng)、神經(jīng)系統(tǒng)、毛囊和性腺等組織也發(fā)現(xiàn)類似的Niche結(jié)構(gòu)[15]。
干細(xì)胞巢是由Niche細(xì)胞、細(xì)胞外基質(zhì)、細(xì)胞因子等構(gòu)成[16],在機(jī)體發(fā)育早期的組織形成和出生之后的組織修復(fù)起重要作用。卵巢生殖干細(xì)胞的周圍存在著調(diào)控其功能的卵巢干細(xì)胞巢,而免疫系統(tǒng)相關(guān)細(xì)胞和分子是卵巢干細(xì)胞巢的重要組成部分。BUKOVSKY[17]指出,在胚胎發(fā)育的早期卵巢干細(xì)胞巢就已經(jīng)形成,由非定位卵巢單核細(xì)胞(MDCs)、T細(xì)胞和血管內(nèi)皮細(xì)胞等組成,而成人卵巢生殖干細(xì)胞巢則由初級MDC(CD14+MDC)、活化的MDC(HLA-DR+MDC)及T細(xì)胞等組成。
卵巢干細(xì)胞巢中的免疫系統(tǒng)相關(guān)的細(xì)胞和分子參與卵巢生殖干細(xì)胞的不對稱分裂,引起生殖細(xì)胞的產(chǎn)生并調(diào)節(jié)其發(fā)生對稱分裂和遷移、生成新的顆粒細(xì)胞及胎兒和成人的原始卵泡、促進(jìn)原始卵泡的選擇和生長以及優(yōu)勢卵泡的形成,從而維持卵巢的正常功能。此結(jié)論基于如下現(xiàn)象[18-20]:(1)卵巢網(wǎng)通道中含有的免疫系統(tǒng)相關(guān)細(xì)胞是卵巢生殖干細(xì)胞形成卵巢所必需的。人類胚胎卵巢是通過生殖嵴區(qū)域腹膜間皮的卵巢干細(xì)胞的進(jìn)化而定性的,由來自于中腎管的卵巢網(wǎng)形成。卵泡的發(fā)育通常始于卵巢皮質(zhì)的最內(nèi)層,靠近卵巢網(wǎng),而卵巢網(wǎng)是卵泡發(fā)育所必需的。卵巢網(wǎng)通道中含有免疫系統(tǒng)相關(guān)的細(xì)胞如小的未提呈的MDC,它們分化成大量活化的MDC,尚有T細(xì)胞。這些免疫系統(tǒng)相關(guān)細(xì)胞是卵巢網(wǎng)形成卵巢必需的。(2)免疫系統(tǒng)相關(guān)細(xì)胞是卵巢生殖干細(xì)胞巢的重要組成部分并觸發(fā)卵巢生殖干細(xì)胞的非對稱分裂。胎兒卵巢生殖干細(xì)胞巢由原始生殖細(xì)胞、未提呈的MDC和T淋巴細(xì)胞組成。未定型的卵巢生殖干細(xì)胞產(chǎn)生于妊娠第6周,先于原始生殖細(xì)胞的到達(dá),原始生殖細(xì)胞在第7周侵入卵巢生殖干細(xì)胞層。卵巢生殖干細(xì)胞只有在細(xì)胞信號如MDC分泌的CD14和T淋巴細(xì)胞分泌的CD8)和激素信號(如絨毛膜促性腺激素和雌二醇)的共同作用下才能發(fā)生非對稱分裂產(chǎn)生次級生殖細(xì)胞,次級生殖細(xì)胞進(jìn)入卵巢皮層,并最終分化為卵母細(xì)胞。被MDC和T細(xì)胞提呈的卵巢生殖干細(xì)胞數(shù)量決定生殖細(xì)胞的數(shù)量。(3)免疫系統(tǒng)相關(guān)細(xì)胞也觸發(fā)卵巢生殖干細(xì)胞的對稱分裂并啟動卵泡的生長,免疫系統(tǒng)的衰退導(dǎo)致卵泡更新的停止。當(dāng)初級CD14+MDC和T淋巴細(xì)胞在一些卵巢表面上皮中擴(kuò)展,觸發(fā)生殖細(xì)胞的對稱分裂;免疫系統(tǒng)的功能在35~40歲左右明顯下降,與此同時,卵泡更新停止。
卵巢衰老是一個多因素相互作用、逐漸累積的復(fù)雜的生理過程,其本質(zhì)是卵巢內(nèi)卵泡數(shù)量的減少和質(zhì)量的降低,表現(xiàn)為生殖功能的喪失和內(nèi)分泌功能的降低[21-22]。女性卵巢衰老的速度要明顯快于機(jī)體其他器官,不僅影響生殖健康,并可引發(fā)嚴(yán)重的圍絕經(jīng)期癥狀和諸多老年性疾病[23],如心血管疾病、癌癥、骨質(zhì)疏松、老年性癡呆、肥胖等。
以往的觀點認(rèn)為機(jī)體和組織器官的衰老是由于干細(xì)胞的衰老所致[24-25],然而近年來,一些學(xué)者指出可能與干細(xì)胞巢的衰老更為緊密[26-27]。在衰老個體中,盡管存在干細(xì)胞,但因為干細(xì)胞巢的不合格會導(dǎo)致各系統(tǒng)的失調(diào)[28]。隨著年齡的增長,卵巢功能并不因為卵巢生殖干細(xì)胞的活動而維持[29]。NIIKURA和其團(tuán)隊發(fā)現(xiàn)減數(shù)分裂特異性標(biāo)志物Stra8和Dazl在無排卵功能老年雌性小鼠卵巢中高度表達(dá)[30],還有學(xué)者在老年鼠、不孕鼠和卵巢早衰鼠的卵巢中均可分離出卵巢生殖干細(xì)胞[31]。這些都說明卵巢功能與卵巢干細(xì)胞的存在與否沒有直接聯(lián)系。此外,把老年小鼠卵巢組織移植進(jìn)年輕小鼠卵巢中,年輕小鼠中會有表達(dá)綠色熒光蛋白(GFP)陽性的原始卵泡形成;但把年輕小鼠卵巢組織移植到老年小鼠卵巢中,則會使年輕小鼠組織中的未成熟卵泡數(shù)目減少[32]。這進(jìn)一步說明卵巢功能的衰老減退主要是由于卵巢生殖干細(xì)胞巢的衰老,而非卵巢生殖干細(xì)胞自身的衰老。
卵巢生殖干細(xì)胞屬于成體干細(xì)胞,目前多數(shù)學(xué)者認(rèn)為引起成體干細(xì)胞衰老的機(jī)制有內(nèi)源性機(jī)制和外源性微環(huán)境兩個方面,主要包括DNA損傷、端??s短、活性氧水平升高、表觀遺傳修飾、細(xì)胞極性改變、代謝及微環(huán)境等方面[33-34],其中外源性微環(huán)境的衰老在引起和加速卵巢生殖衰老中起重要的作用。而干細(xì)胞巢由血管間分隔室組成,血管間分隔室把干細(xì)胞與免疫系統(tǒng)、循環(huán)系統(tǒng)連接起來了[35]。因此免疫系統(tǒng)和循環(huán)系統(tǒng)的功能改變可能會對卵巢干細(xì)胞巢的衰老產(chǎn)生影響。
對于循環(huán)系統(tǒng),JOHNSON等[36]提出成年小鼠骨髓及外周血中存在一種干細(xì)胞,可對雌性哺乳動物的卵泡池進(jìn)行補(bǔ)充。但學(xué)者們紛紛提出質(zhì)疑,EGGAN等[37]通過循環(huán)系統(tǒng)相連的小鼠研究,并沒有發(fā)現(xiàn)卵細(xì)胞嵌合的現(xiàn)象。此外,對模擬放療造模的小鼠進(jìn)行骨髓移植,移植后的小鼠也并不能產(chǎn)生卵細(xì)胞。因此EGGAN等[37]的理論推翻了JOHNSON等提出的理論。LEE等[38]指出骨髓移植后對模擬放療造模處理而長期不孕的小鼠的生殖功能有拯救作用,但其后代均來源于受體小鼠生殖系。之后又有學(xué)者指出骨髓移植對化療藥物引起的卵巢功能損傷具有修復(fù)作用[39-40]。近年來,有學(xué)者研究發(fā)現(xiàn)卵巢干細(xì)胞總是伴隨著非常小的胚胎干細(xì)胞存在于成年哺乳動物卵巢內(nèi)[41-42],而且人類非常小的胚胎干細(xì)胞在年輕、中年和老年人的正常外周血中均存在[43]。VIRANTKLUN等[44]經(jīng)研究指出VSELs與生發(fā)血統(tǒng)和造血之間存在潛在聯(lián)系。VAN等[45]發(fā)現(xiàn)“同伴”血細(xì)胞對于卵巢生殖干細(xì)胞巢的形成和穩(wěn)態(tài)是必不可少的。由此可以看出循環(huán)系統(tǒng)對于卵巢功能的提升至關(guān)重要。
免疫系統(tǒng)與卵巢功能的關(guān)系在很早就已被發(fā)現(xiàn),但其對卵巢生殖干細(xì)胞巢影響未引起研究者的重視。SAKAKVRA等[46]摘除2~4 d的新生小鼠胸腺后發(fā)現(xiàn)該類小鼠體內(nèi)出現(xiàn)卵巢發(fā)育不全的現(xiàn)象;RUSSELL等[47]用正常雌鼠的胸腺細(xì)胞可使小鼠體內(nèi)經(jīng)環(huán)磷酰胺和X射線誘導(dǎo)的超排卵現(xiàn)象得到抑制;1979年,BUKOVSKY等[48]首先提出免疫系統(tǒng)在調(diào)節(jié)卵巢功能中發(fā)揮重要作用。近年來,有學(xué)者發(fā)現(xiàn)給予新生裸鼠肌肉注射胸腺素,該類小鼠發(fā)育到成年期時血清中的卵泡生長素和黃體生成素可維持穩(wěn)定水平,并且使裸鼠卵巢發(fā)育不全現(xiàn)象的發(fā)生得到抑制[49];此外,研究者還發(fā)現(xiàn),隨著年齡的增長,機(jī)體免疫系統(tǒng)不可避免的發(fā)生退化,這種退化首先表現(xiàn)為胸腺功能受損,其次就是卵巢功能的衰退。這些研究結(jié)果證明,機(jī)體免疫系統(tǒng)與卵巢功能之間存在著密切聯(lián)系,但免疫系統(tǒng)的衰老使卵巢生殖干細(xì)胞巢發(fā)生了哪些變化及其具體的機(jī)制目前還有待于進(jìn)一步研究。
對于卵巢早衰和圍絕經(jīng)期患者,可以通過飲食、運(yùn)動、保健品等提升自身的免疫功能和改善循環(huán)系統(tǒng)功能,從而間接為正在衰老的卵巢提供了一個適宜的微環(huán)境,使得卵巢干細(xì)胞可以繼續(xù)保持活性,繼續(xù)填補(bǔ)卵泡池,延緩了女性生殖衰老,也進(jìn)而改善了伴隨卵巢衰老帶來的圍絕經(jīng)期癥狀和諸多的老年性疾病。
對于成年女性,癌癥治療后導(dǎo)致的不育可先在卵細(xì)胞庫凍存卵細(xì)胞,在通過輔助生殖技術(shù)補(bǔ)救。然而這種方法對于青春期前的患者不適用,因為卵細(xì)胞發(fā)生還未開始。對于這些患者,在治療前,可以切除部分卵巢組織,然后消化為組織懸液或單個細(xì)胞懸液凍存起來。當(dāng)需要時再把之前凍存的懸液移植到殘留的卵巢中,當(dāng)進(jìn)入青春期后便又可恢復(fù)卵巢功能。
衰老與干細(xì)胞衰老密切相關(guān)。在卵巢衰老中,卵巢干細(xì)胞巢的衰老和退化被認(rèn)為是導(dǎo)致卵巢生殖功能衰退的主要因素,而并非卵巢生殖干細(xì)胞本身的衰老。免疫系統(tǒng)和循環(huán)系統(tǒng)對卵巢干細(xì)胞巢的衰老有至關(guān)重要的作用,但其具體機(jī)制還需進(jìn)一步研究。當(dāng)前,環(huán)境污染、化學(xué)殘留、職業(yè)暴露、生活壓力以及醫(yī)療風(fēng)險等因素對女性卵巢功能造成的損害,使不孕癥及卵巢功能早衰已成為影響人類繁衍與女性身心健康的主要疾病。據(jù)統(tǒng)計,目前全球8 000萬~1.1億不孕癥患者中,約40%女性與卵巢功能衰退有關(guān),約1%而未及40歲的婦女因罹患卵巢早衰而提前絕經(jīng);另一方面,由于人類平均壽命延長以及二胎政策的放開,受生理性卵巢衰老所困的高齡女性對于提高生活質(zhì)量以及延長生殖壽命的需求日益強(qiáng)烈。因此,探討卵巢生殖衰老發(fā)生的機(jī)理并通過提高免疫力從而間接延緩卵巢衰老過程的發(fā)生具有重要意義及廣泛前景。
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