余志鑫，李鵬宇，李凱，繆時(shí)英，王琳芳，宋偉

綜 述

精原干細(xì)胞微環(huán)境研究進(jìn)展

余志鑫，李鵬宇，李凱，繆時(shí)英，王琳芳，宋偉

中國(guó)醫(yī)學(xué)科學(xué)院基礎(chǔ)醫(yī)學(xué)研究所，北京協(xié)和醫(yī)學(xué)院基礎(chǔ)學(xué)院，醫(yī)學(xué)分子生物學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室，北京 100005

精原干細(xì)胞(spermatogonia stem cells, SSCs)是一類(lèi)在睪丸中具有長(zhǎng)期自我更新和分化潛能的生殖細(xì)胞(germ cells, GCs)，即位于基底膜上的組織干細(xì)胞，其自我更新和分化受到周?chē)h(huán)境的調(diào)控。近年來(lái)對(duì)SSCs的研究取得了一系列重要進(jìn)展，為臨床治療部分男性不育患者帶來(lái)了曙光。其中，微環(huán)境對(duì)SSCs的調(diào)節(jié)功能的研究尤為重要，微環(huán)境負(fù)責(zé)整合不同類(lèi)型的細(xì)胞成分、細(xì)胞外基質(zhì)、細(xì)胞外調(diào)節(jié)分子及激素等對(duì)SSCs的作用，從而調(diào)節(jié)SSCs命運(yùn)。關(guān)于SSCs微環(huán)境的研究已開(kāi)始逐步成為干細(xì)胞研究的主要內(nèi)容之一。本文主要對(duì)小鼠()SSCs微環(huán)境的細(xì)胞組成、調(diào)控因子以及特點(diǎn)等研究現(xiàn)狀進(jìn)行了綜述，為深入研究SSCs微環(huán)境的結(jié)構(gòu)和功能提供背景資料，希望在未來(lái)能夠通過(guò)多種研究模式復(fù)用，發(fā)現(xiàn)更為豐富的細(xì)胞表型和微環(huán)境因子。

精原干細(xì)胞；微環(huán)境；自我更新；分化；激素調(diào)控

精原干細(xì)胞(spermatogonia stem cells, SSCs)位于曲細(xì)精管的基底膜上，對(duì)于精子發(fā)生(spermato-genesis)以及所有物種的繁殖都至關(guān)重要，并始終保持著自我更新和定向分化能力，一部分SSCs參與精子發(fā)生，而另一部分形成了SSC池。SSCs自我更新和分化之間的協(xié)調(diào)保證了SSCs在體內(nèi)保持動(dòng)態(tài)平衡，其減少或異常增殖都可能影響精子發(fā)生，從而導(dǎo)致男性不育。SSCs的自我更新和分化受到內(nèi)在基因表達(dá)和外在環(huán)境的精確調(diào)控，因此對(duì)其調(diào)控機(jī)制的研究對(duì)于治療男性不育具有重要意義。SSCs與體細(xì)胞以及體細(xì)胞分泌的多種因子之間相互作用構(gòu)成了可持續(xù)產(chǎn)生精子的環(huán)境，稱(chēng)為微環(huán)境，也稱(chēng)為“niche”[1]。在此之前，支持細(xì)胞(sertoli cells, SCs)被認(rèn)為是微環(huán)境的主要組成部分，近年來(lái)隨著研究的深入，發(fā)現(xiàn)多種細(xì)胞類(lèi)型和因子也參與微環(huán)境的組成，且SSCs的自我更新和分化與微環(huán)境中各類(lèi)細(xì)胞的數(shù)量、生長(zhǎng)因子及其下游信號(hào)通路、多種轉(zhuǎn)錄因子和表觀(guān)調(diào)控因子等密切相關(guān)。微環(huán)境的研究為臨床治療男性不育癥以及體外培養(yǎng)SSCs提供了新思路。本文總結(jié)了近幾年關(guān)于SSCs微環(huán)境的組成和調(diào)節(jié)最新研究進(jìn)展，以期為SSCs微環(huán)境的深入研究提供參考。

1 SSCs及其微環(huán)境

1.1 SSCs概述

未分化精原細(xì)胞(undifferentiated spermatogonia)約占成年小鼠()睪丸生殖細(xì)胞(germ cells, GCs)總數(shù)的0.3%，恒河猴()為4%，人類(lèi)()為22%[2～4]。小鼠精子發(fā)生過(guò)程包括未分化精原細(xì)胞As (A single)、Apr (A paired)和Aal (A aligned)，分化中的精原細(xì)胞(differentiating spermatogonia)A1依次形成A2、A3、A4、In型和B型精原細(xì)胞，再經(jīng)過(guò)減數(shù)分裂形成初級(jí)精母細(xì)胞、次級(jí)精母細(xì)胞、圓形精子細(xì)胞，最終變形為成熟精子(圖1)[5]。一般認(rèn)為SSCs是As型精原細(xì)胞，且成年小鼠睪丸中大約有35,000個(gè)As細(xì)胞，而GFRα1陽(yáng)性的As更接近真正意義上的SSCs[6～8]。在靈長(zhǎng)類(lèi)動(dòng)物的基底膜上存在Ad(A dark)型和Ap(A pale)型未分化精原細(xì)胞以及B型分化型精原細(xì)胞，通常認(rèn)為Ap是激活型的精原細(xì)胞，Ad是靜止型精原細(xì)胞即SSCs[5,9]。SSCs的能力取決于它們產(chǎn)生兩種后代的能力：一種復(fù)制母干細(xì)胞(自我更新)，另一種獲得特殊的形態(tài)和功能成為精子(分化)[10]。SSCs在微環(huán)境中保持自我更新和分化之間的平衡是通過(guò)SSCs細(xì)胞群體的不對(duì)稱(chēng)分裂，而不是通過(guò)恒定的單個(gè)細(xì)胞對(duì)稱(chēng)分裂所實(shí)現(xiàn)的[5,11]。在SSCs中除了確實(shí)進(jìn)行自我更新的“實(shí)際干細(xì)胞”之外，還存在第二個(gè)群體—“潛在干細(xì)胞”，其在正常情況下不會(huì)自我更新，僅僅在損傷發(fā)生后啟動(dòng)其潛能[12]。

1.2 微環(huán)境定義

SSCs微環(huán)境被認(rèn)為是一個(gè)開(kāi)放的系統(tǒng)，不能單純地從解剖學(xué)上定義，而應(yīng)該從分子生物學(xué)上定義。微環(huán)境最初是為了解釋造血干細(xì)胞移植后的行為[13,14]。而在精子發(fā)生中的微環(huán)境有狹義和廣義之分：廣義的微環(huán)境主要包括大部分的睪丸體細(xì)胞和其所分泌的因子和激素；狹義的微環(huán)境大多僅指與SSCs直接相鄰的體細(xì)胞所構(gòu)成的微環(huán)境[15,16]。在成年睪丸組織中，SSCs大多數(shù)位于曲細(xì)精管的基底膜上，幾乎由SCs完全包繞，周?chē)w細(xì)胞及其合成分泌的各種細(xì)胞因子共同構(gòu)成了精子發(fā)生的微環(huán)境。這些生長(zhǎng)因子和細(xì)胞外信號(hào)決定了SSCs的命運(yùn)，最終在小鼠中經(jīng)過(guò)35天形成成熟精子，在人類(lèi)中經(jīng)過(guò)64天形成成熟精子[17]。在小鼠中其生精上皮周期分為12個(gè)時(shí)期，其中最為特殊的是VII～VIII期，是精原細(xì)胞分化的時(shí)期[18～20]。本文中的微環(huán)境則指廣義上的開(kāi)放的微環(huán)境。

1.3 SSCs微環(huán)境的基本特征

在正常組織中，Yoshida等[21,22]研究表明SSCs優(yōu)先定位在與間質(zhì)組織和脈管系統(tǒng)相鄰的管狀區(qū)域，對(duì)于FGFs的競(jìng)爭(zhēng)調(diào)節(jié)了小管基底層內(nèi)SSCs種群的密度和大小。關(guān)于SSCs微環(huán)境的分布存在爭(zhēng)議：如Oatley等[23]證明決定微環(huán)境分布的是SCs的數(shù)量而不是血管的位置；Brett Nixon等[24]則認(rèn)為SSCs定位在缺血區(qū)，主要通過(guò)糖酵解供能，而祖細(xì)胞和分化生殖細(xì)胞優(yōu)先定位在與脈管系統(tǒng)相鄰的區(qū)域，以得到充足的氧氣進(jìn)行氧化磷酸化(oxidative phosph-orylation, OXPHOS)，產(chǎn)生豐富的ATP來(lái)推動(dòng)快速擴(kuò)增分裂；Mohyeldin等[25]認(rèn)為低氧可維持多種干細(xì)胞表型、減少增殖和未分化狀態(tài)。總之，關(guān)于SSCs所處的位置及代謝方式還有待進(jìn)一步解決。

圖1 小鼠精子發(fā)生過(guò)程示意圖

小鼠精子發(fā)生過(guò)程：未分化精原細(xì)胞(A undiff)由A single、A paired、A al-4、A al-8和A al-16相互連接的細(xì)胞組成，逐漸分化為A1、A2、A3、A4、In型和B型，B型精原細(xì)胞經(jīng)減數(shù)分裂成精母細(xì)胞，最終產(chǎn)生精子。

2 SSCs微環(huán)境的細(xì)胞組成

2.1 支持細(xì)胞

SCs是唯一與GCs直接接觸的體細(xì)胞，在其基部與周?chē)鶶Cs和生精細(xì)胞相連接，每個(gè)SC可支持30～50個(gè)不同發(fā)育階段的GCs，為精子發(fā)生提供物理支持和穩(wěn)定微環(huán)境[26]。SCs可通過(guò)分泌多種生長(zhǎng)因子如膠質(zhì)細(xì)胞源性神經(jīng)營(yíng)養(yǎng)因子(glial cell line- derived neurotrophic factor, GDNF)、維甲酸(retinoic acid, RA)、干細(xì)胞因子(stem cell factor, SCF)、成纖維細(xì)胞生長(zhǎng)因子2(fibroblast growth factor-2, FGF2)以及激活素調(diào)節(jié)SSCs自我更新和分化；也可通過(guò)緊密連接(tight junctions, TJs)參與血睪屏障(blood- testis barrier, BTB)的形成，保護(hù)生精細(xì)胞免受自身免疫系統(tǒng)的干擾，以多種方式支持、培育和保護(hù)GCs；另外其還可以表達(dá)自殺配體相關(guān)因子(factor associated suicide ligand, FasL)和轉(zhuǎn)化生長(zhǎng)因子-β (transforming growth factor-β, TGF-β)等蛋白，有助于局部免疫豁免；SCs也可吞噬、消化精子發(fā)生過(guò)程中脫落的殘余胞質(zhì)[5,27,28]。SCs形成的BTB在精子發(fā)生的減數(shù)分裂中起到重要作用，Cldn11是BTB主要成份，其缺失后SSCs不能有效定植，精子發(fā)生阻滯停留在精母細(xì)胞階段最終導(dǎo)致不育[29]。BTB是一種高度選擇性的屏障，包括緊密連接、橋粒樣連接和間隙連接，移植實(shí)驗(yàn)中移植1周后SSCs才能穿過(guò)BTB完全遷移到基底膜[30]。此外，SCs除了支持精子發(fā)生外，也可以保留正常的間質(zhì)細(xì)胞群并支持正常的管周肌樣細(xì)胞(peritubular myoid cells, PMCs)的功能[31]。

2.2 管周肌樣細(xì)胞

曲細(xì)精管被PMCs包裹，與SCs共同形成生精上皮基膜。PMCs主要在間質(zhì)細(xì)胞分泌的激素作用下收縮，也可以在GATA4的作用下抑制收縮[32]。除了提供結(jié)構(gòu)支持和推動(dòng)內(nèi)容物流向睪丸網(wǎng)外，PMCs還分泌對(duì)SSCs重要的旁分泌因子，包括GDNF和巨噬細(xì)胞集落刺激因子-1 (macrophage colony stimu-lating factor-1, CSF-1)。以往的研究大多側(cè)重于SCs所分泌的GDNF，但近幾年的研究表明PMCs產(chǎn)生的GDNF對(duì)小鼠和人的SSCs維持也至關(guān)重要[6,33,34]。PMCs同時(shí)也參與雄激素受體(androgen receptor, AR)的表達(dá)，PMCs中AR的特異性敲除可導(dǎo)致睪丸重量顯著減輕，無(wú)精子發(fā)生，所有類(lèi)型GCs數(shù)量減少[35]。

2.3 睪丸內(nèi)皮細(xì)胞

睪丸內(nèi)皮細(xì)胞(testicular endothelial cells, TECs)是干細(xì)胞生物學(xué)中細(xì)胞因子的豐富來(lái)源，F(xiàn)GF2可促進(jìn)TECs產(chǎn)生GDNF，且相比于SCs，TECs是GDNF的主要來(lái)源[36]。TECs除產(chǎn)生GDNF外，它也可以產(chǎn)生FGF2、基質(zhì)細(xì)胞衍生因子-1 (stromal cell derived factor-1, SDF-1)、巨噬細(xì)胞炎癥蛋白2 (macrophage inflammatory protein-2, MIP-2)、胰島素樣生長(zhǎng)因子結(jié)合蛋白2 (insulin-like growth factor binding protein-2, IGFBP-2)以及一氧化氮(nitric oxide, NO)，其中NO可抑制間質(zhì)細(xì)胞(leydig cells, LCs)類(lèi)固醇激素的產(chǎn)生[37]。最近研究表明，TECs能夠在沒(méi)有外源性GDNF的情況下在體外支持SSCs，并且其衍生因子顯著促進(jìn)細(xì)胞毒性損傷后生精上皮的重新填充[36]。此外，在大鼠()中，TECs可作為飼養(yǎng)層細(xì)胞，增強(qiáng)SSCs的增殖和自我更新的能力，同時(shí)保持其干性[38]。這些數(shù)據(jù)表明，TECs是SSCs生態(tài)位的關(guān)鍵組成部分。

2.4 睪丸間質(zhì)細(xì)胞

睪丸LCs分泌的睪酮(testosterone)是睪丸中主要的旁分泌因子，雄激素與其AR結(jié)合后介導(dǎo)生精細(xì)胞的信號(hào)通路[39]。通過(guò)對(duì)發(fā)育中的人類(lèi)睪丸細(xì)胞進(jìn)行單細(xì)胞分析，發(fā)現(xiàn)LCs和SCs起源于同一個(gè)異質(zhì)祖細(xì)胞亞群，這表明睪丸LCs和SCs具有同源性[40]。除睪酮外，LCs還可分泌眾多激素如肽類(lèi)激素抗利尿激素、催產(chǎn)素和前列腺素等對(duì)PMCs的收縮起重要作用[41,42]。LCs也可以分泌胰島素樣生長(zhǎng)因子-1 (insulin-like growth factor-1, IGF-1)和CSF-1[43～45]。但其主要功能還是合成雄激素，男性體內(nèi)95%的雄激素是由LCs合成及分泌。

2.5 睪丸巨噬細(xì)胞

在男性生殖器官中，睪丸和附睪是兩個(gè)獨(dú)特的免疫部位。睪丸巨噬細(xì)胞(testicular macrophages, TMs)大約占睪丸間質(zhì)細(xì)胞的20%，在睪丸中分為間質(zhì)巨噬細(xì)胞和管周巨噬細(xì)胞[46]。間質(zhì)巨噬細(xì)胞來(lái)源于胚胎的卵黃囊細(xì)胞，但在出生后被骨髓來(lái)源的巨噬細(xì)胞取代；管周巨噬細(xì)胞在出生后出現(xiàn)且來(lái)源于骨髓中單核細(xì)胞[47,48]。近年來(lái)研究發(fā)現(xiàn)，睪丸中存在獨(dú)立于單核細(xì)胞的TMs，即骨髓來(lái)源的單核細(xì)胞對(duì)于TMs池的補(bǔ)充無(wú)實(shí)質(zhì)性貢獻(xiàn)，但通過(guò)流式細(xì)胞分選等方法發(fā)現(xiàn)睪丸白細(xì)胞主要來(lái)源于單核吞噬細(xì)胞[49～52]。而TMs對(duì)SSCs的自我更新和分化的影響直到2015年才被關(guān)注，研究發(fā)現(xiàn)TMs可產(chǎn)生CSF-1并表達(dá)RA合成的酶，且可能參與調(diào)控SSCs的維持和分化[47,53]。

2.6 其他細(xì)胞群

除上述主要細(xì)胞群外，目前被廣泛研究的還包括淋巴管內(nèi)皮細(xì)胞(lymphatic endothelial cells, LECs)。LECs位于生精小管和睪丸間質(zhì)的邊緣，覆蓋淋巴間隙的表面，靠近脈管系統(tǒng)的LECs可表達(dá)大量FGFs[21]。隨著單細(xì)胞測(cè)序等技術(shù)的發(fā)展，逐漸有新的亞群被發(fā)現(xiàn)，關(guān)于睪丸細(xì)胞的分群也逐漸精細(xì)。例如：2018年Wang等[54]將人類(lèi)睪丸細(xì)胞分為17個(gè)群，其中14個(gè)為GCs，另外3個(gè)為體細(xì)胞；同年，小鼠睪丸GCs被分為4個(gè)群，包括精原細(xì)胞、精母細(xì)胞、圓形精子細(xì)胞和長(zhǎng)形精子細(xì)胞，體細(xì)胞被分為7個(gè)群，包括SCs、LCs、PMCs、TECs、先天性淋巴細(xì)胞(innate lymphoid, IL)、睪丸巨噬細(xì)胞以及未知細(xì)胞群[55]；2022年，大鼠精原細(xì)胞被分為5個(gè)群，在原來(lái)的基礎(chǔ)上新增了凝聚型精子(condensed spermatids, CSPT)[56]。

3 SSCs微環(huán)境的調(diào)控因子

在SSCs微環(huán)境中有多種因子調(diào)節(jié)其自我更新或分化，例如已知的GDNF、FGF2、CSF-1、白血病抑制因子(leukemia inhibitory factor, LIF)和IGF-1等調(diào)節(jié)SSCs的自我更新[5]。而RA、骨形態(tài)發(fā)生蛋白4 (bone morphogenetic protein4, BMP4)和SCF等可促進(jìn)SSCs的分化(圖2)[5]。促進(jìn)自我更新和分化的細(xì)胞外微環(huán)境雖然并不在空間上產(chǎn)生分隔，但GDNF和RA等這些因素顯示出不同時(shí)間的時(shí)間波動(dòng)，隨生精上皮周期的變化而變化，這預(yù)示著SSCs和分化細(xì)胞很可能暴露于相同的細(xì)胞外信號(hào)[17,57,58]。

圖2 SSCs微環(huán)境細(xì)胞組成及調(diào)控因子

SSCs微環(huán)境細(xì)胞組成及調(diào)控因子：SCs可產(chǎn)生GDNF、RA、SCF和FGF2等，PMCs可產(chǎn)生GDNF和CSF-1等，TECs可產(chǎn)生GDNF和FGF2等，LCs可產(chǎn)生IGF-1、RA和CSF-1等，這些因子與細(xì)胞相互作用共同構(gòu)成微環(huán)境。

3.1 參與自我更新的調(diào)控因子

3.1.1 膠質(zhì)細(xì)胞源性神經(jīng)營(yíng)養(yǎng)因子

GDNF在睪丸中主要由SCs和TECs分泌，其次是已分化生殖細(xì)胞、PMCs和LCs。GDNF對(duì)精原細(xì)胞的增殖影響有劑量依賴(lài)：在過(guò)表達(dá)GDNF的小鼠中，未分化精原細(xì)胞聚集在生精小管中并停止分化，而在GDNF雜合小鼠中，精子發(fā)生伴隨著精原細(xì)胞的丟失而逐漸停滯[59～61]。GDNF可以通過(guò)GFRα1/c-Ret兩種受體促進(jìn)SSCs的自我更新[62,63]。GFRα1和RET敲除也表現(xiàn)出與上述GDNF敲除 同樣的現(xiàn)象，即由于精原細(xì)胞丟失精子發(fā)生逐漸停滯[64,65]。同樣，PMCs中基因的破壞也能影響未分化精原細(xì)胞發(fā)育[6]。在GDNF、FGF2、EGF和LIF存在的培養(yǎng)基中培養(yǎng)SSCs，可生長(zhǎng)2年，且在小鼠胚胎成纖維細(xì)胞(mouse embryo fibroblasts, MEFs)上呈葡萄狀生長(zhǎng)[17,66,67]。有研究發(fā)現(xiàn)，在精子發(fā)生的穩(wěn)定階段和白消安(busulfan)介導(dǎo)的精原細(xì)胞損傷后恢復(fù)階段，盡管SSCs數(shù)量增加但GDNF是通過(guò)阻斷分化而不是促進(jìn)增殖來(lái)促進(jìn)自我更新[57]。最近又有研究表明SSCs的數(shù)量因GDNF信號(hào)傳導(dǎo)的瞬時(shí)抑制而減少，但在信號(hào)傳導(dǎo)恢復(fù)后通過(guò)自我更新復(fù)制恢復(fù)[68]。

3.1.2 成纖維細(xì)胞生長(zhǎng)因子2

FGF2由SCs分泌，對(duì)SSCs的自我更新起促進(jìn)作用。FGF的靶基因包括、、和，受體包括FGFR1、FGFR2、FGFR3和FGFR4，主要靶細(xì)胞為未分化精原細(xì)胞[21]。有研究發(fā)現(xiàn)，GDNF和FGF2都增加了未分化精原細(xì)胞中GFRα1陽(yáng)性細(xì)胞的亞群，但FGF2擴(kuò)增亞群表達(dá)RARγ的頻率是GDNF的1.9倍，即FGF2擴(kuò)大了一個(gè)易于分化的亞群[61,69]。FGF2能刺激TECs表達(dá)GNDF，從而間接調(diào)控SSCs的數(shù)量恒定[36]。未分化型精原細(xì)胞可以在FGF2存在而無(wú)GDNF條件下在體外維持，但在該條件下的細(xì)胞顯示出生長(zhǎng)不良和干性降低。

3.1.3 巨噬細(xì)胞集落刺激因子1

CSF-1也稱(chēng)為MCSF-1，在小鼠睪丸中主要由TMs、LCs、PMCs和精原細(xì)胞產(chǎn)生，其受體CSF1R主要表達(dá)于GFRα1陽(yáng)性和THY1陽(yáng)性的精原細(xì)胞中[44,70]。近年來(lái)研究發(fā)現(xiàn)CSF1R在小鼠TMs、LCs、SCs和PMCs等細(xì)胞中表達(dá)，且CSF1R不僅是CSF的受體，也是IL-34的受體[70,71]。CSF-1蛋白在1周內(nèi)的小鼠睪丸中表達(dá)最高，并隨年齡顯著降低，其可增強(qiáng)的配體作用，促進(jìn)精子發(fā)生直至精子產(chǎn)生[72]。研究表明，CSF-1可以促進(jìn)SSCs的增殖和自我更新[44,73]。β-雌二醇以劑量和時(shí)間依賴(lài)方式促進(jìn)SCs分泌CSF-1進(jìn)而促進(jìn)人類(lèi)胎兒SSCs的增殖并抑制SSCs凋亡，然而與雌激素受體抑制劑結(jié)合又可以完全逆轉(zhuǎn)其對(duì)抑制SSCs凋亡的作用[74]。

3.1.4 趨化因子CXC模體12

趨化因子CXC模體12 (C-X-C motif chemokine 12，CXCL12)在人中由LCs分泌，在小鼠中由SCs分泌，并與SSCs上的CXCR4受體結(jié)合以促進(jìn)SSCs的自我更新和維持，尤其是CXCL12-CXCR4信號(hào)通路可促進(jìn)SSCs增殖并阻斷RA誘導(dǎo)SSCs分化[15,75]。有研究表明CXCL12、GDNF和FGF2已形成了影響SSCs自我更新的調(diào)控網(wǎng)絡(luò)，GDNF可上調(diào)CXCL12受體的表達(dá)。此外，CXCR4在胚胎發(fā)生期間參與原始生殖細(xì)胞(primordial germ cells, PGCs)歸巢，在出生后的睪丸中也參與SSCs歸巢至生態(tài)位，CXCL12轉(zhuǎn)染SCs可提高歸巢效率[76,77]。

3.1.5 白血病抑制因子

LIF主要由PMCs產(chǎn)生，其受體LIFR在小鼠的所有GCs、SCs、LCs和TMs中均表達(dá)，且SCs中的LIFR最為重要[78～80]。LIF在體外可增強(qiáng)新生小鼠睪丸培養(yǎng)中GCs集落的形成，目前報(bào)道最多的是在培養(yǎng)基中添加bFGF、GDNF、LIF和EGF使SSCs在體外迅速增殖[81～84]。在使用明膠和含有GDNF、FGF2、LIF和EGF的培養(yǎng)基中，6天后也產(chǎn)生了很大比例的雄性人類(lèi)胎兒生殖細(xì)胞，這表明體外培養(yǎng)人GCs又前進(jìn)了一步[85]。

3.1.6 活性氧

除上述調(diào)控因子外，微環(huán)境中還存在其他因素，如活性氧(reactive oxygen species, ROS)。一直以來(lái)普遍認(rèn)為ROS對(duì)干細(xì)胞有害，近年來(lái)首次證明了ROS介導(dǎo)的正反饋在維持SSCs自我更新方面起著重要作用[86]。ROS在GDNF和FGF2存在狀態(tài)下由SSCs中的NADPH氧化酶3 (NADPH oxidase 3, NOX3)產(chǎn)生，在穩(wěn)定增殖階段由SSCs中的NADPH氧化酶1(NADPH oxidase 1, NOX1)產(chǎn)生，兩種NOX在不同條件下調(diào)節(jié)SSCs中ROS的產(chǎn)生[87]。添加過(guò)氧化氫(hydrogen peroxide, H2O2)使ROS含量升高時(shí)，可明顯促進(jìn)干細(xì)胞的增殖，而NOX1的缺乏又抑制SSCs的自我更新[88]。另外NOX1通過(guò)ROS-BCL6B-NOX1途徑產(chǎn)生的ROS和氧氣相互作用共同決定SSCs的自我更新效率[89]。這些研究都表明ROS在SSCs自我更新方面起了重要作用。

3.1.7 其他

除上述因子外，在微環(huán)境中還存在許多因素影響SSCs的自我更新。CARF (collaborator of ARF)可以通過(guò)Wnt信號(hào)通路促進(jìn)SSCs的自我更新和增殖[90]。FGF9由睪丸中的體細(xì)胞產(chǎn)生，是SSCs增殖的重要調(diào)節(jié)因子，另外有研究表明，由LECs產(chǎn)生的FGFs的其他成員如FGF4、FGF5和FGF8都可以調(diào)節(jié)SSCs的數(shù)目[21,91]。最近又發(fā)現(xiàn)一種新的因子—白細(xì)胞介素-34 (interleukin-34, IL-34)，它是一種新型的旁分泌/自分泌因子，可以在LCs、SCs和部分精原細(xì)胞中表達(dá)以調(diào)節(jié)精子發(fā)生[92]。上述因子都在SSCs自我更新中起著重要作用，其他新的因子還有待進(jìn)一步研究發(fā)現(xiàn)。

3.2 參與分化的調(diào)控因子

3.2.1 維甲酸

RA是維生素A的中間代謝產(chǎn)物，主要在SCs、GCs、PMCs和LCs中表達(dá)， RALDH1和RALDH2分別是SCs和GCs中的關(guān)鍵RA合成酶[93,94]。SCs產(chǎn)生的RA及其靶基因在小鼠中可調(diào)節(jié)精原細(xì)胞分化的啟動(dòng)，周期性RA-信號(hào)傳導(dǎo)與周期性GCs能力相互協(xié)調(diào)，以調(diào)節(jié)兩種不同的細(xì)胞類(lèi)型特異性反應(yīng)：SSCs精子分化和減數(shù)分裂起始[61,95]。雌性GCs在性別決定后不久開(kāi)始減數(shù)分裂，而雄性GCs進(jìn)入靜止?fàn)顟B(tài)，則是因?yàn)樗鼈冎車(chē)腟Cs表達(dá)RA代謝酶，如CYP26B1，在RA作用于GCs之前有效降解RA[96]。精原細(xì)胞的分化依賴(lài)于生精上皮內(nèi)部合成的RA，然而，在完全沒(méi)有RA的情況下，精原細(xì)胞也可以啟動(dòng)減數(shù)分裂并表達(dá)[93,97]。在體外，RA與SCs配合或單獨(dú)作用均能在不同程度上引起不同年齡段小鼠GCs的分化，且其誘導(dǎo)分化傾向與精原細(xì)胞鏈的長(zhǎng)度有關(guān)[98]。近來(lái)有研究表明，RA與孕酮在體外協(xié)同作用于小鼠誘導(dǎo)多功能干細(xì)胞分化為雄性GCs[99]。

3.2.2 骨形態(tài)發(fā)生蛋白4

BMP4屬于TGF-β家族，主要在GCs中表達(dá)，其表達(dá)水平隨著精子發(fā)生的進(jìn)行而升高，其受體BMPR1A和BMPRII存在于精原細(xì)胞、精母細(xì)胞和圓形精子中，在精子發(fā)生過(guò)程中起重要作用[100]。在BMP4存在條件下SSCs更傾向分化，在大鼠中BMP4不但可以誘導(dǎo)SSCs分化，同時(shí)可以導(dǎo)致細(xì)胞的黏附性發(fā)生改變，如細(xì)胞黏附分子和肌動(dòng)蛋白細(xì)胞骨架調(diào)節(jié)途徑等[101]。此外BMP4可通過(guò)上調(diào)Sohlh2的表達(dá)，在SSCs的早期分化中發(fā)揮重要作用。BMP4及其多種受體存在于人SCs中，細(xì)胞增殖和Brdu摻入測(cè)定表明BMP4能促進(jìn)SCs的DNA合成和復(fù)制[102]。另外BMP4也能與RA協(xié)同作用，誘導(dǎo)體內(nèi)精原細(xì)胞分化[103]。

3.2.3 干細(xì)胞因子

SCF在哺乳動(dòng)物睪丸中由SCs產(chǎn)生[104]。有研究表明，卵泡刺激素(follicle-stimulating hormone, FSH)可通過(guò)SCF/C-Kit信號(hào)通路調(diào)節(jié)SCF的表達(dá)，在FSH的刺激下，SCF會(huì)急劇升高[105]。通過(guò)與C-kit受體結(jié)合來(lái)觸發(fā)其生物學(xué)功能，它們組成SCF/C-kit系統(tǒng)在雄性和雌性生殖道內(nèi)發(fā)揮重要作用，近年來(lái)關(guān)于SCF/C-kit異常表達(dá)引起雄性生殖系統(tǒng)腫瘤和雌性生殖系統(tǒng)腫瘤的研究日益深入[106～108]。SCF通過(guò)轉(zhuǎn)錄上調(diào)、、、和基因改善SSCs的體外分化，并對(duì)表達(dá)C-kit的精原細(xì)胞的有絲分裂和存活的影響依賴(lài)于不同的信號(hào)通路[109～111]。有研究表明人重組SCF (recombinant human SCF, rhSCF)可促進(jìn)蠑螈()精子的增殖，但不能促進(jìn)減數(shù)分裂的開(kāi)始[112]。

3.2.4 其他

除上述因子外，還存在諸多因子影響SSCs的分化。近年來(lái)發(fā)現(xiàn)的泛素羧基末端水解酶-L1 (ubiq-uitin carboxy-terminal hydrolase-l1, UCH-L1)，其缺失可降低SSCs的分化能力，并影響SSCs穩(wěn)態(tài)和新陳代謝的維持[113]。這些分化因子可能與其他因子，如自我更新因子和分化因子協(xié)同作用調(diào)節(jié)SSCs的自我更新和分化。

3.3 參與精子發(fā)生的轉(zhuǎn)錄因子調(diào)控

微環(huán)境中的一些轉(zhuǎn)錄因子也會(huì)對(duì)SSCs產(chǎn)生影響，例如影響SSCs自我更新相關(guān)的轉(zhuǎn)錄因子Plzf、Bcl6b、Etv5、ID4和Foxo1等，以及促進(jìn)SSCs分化的相關(guān)轉(zhuǎn)錄因子如Sall4以及Sohlh1/2等。近年來(lái)對(duì)SSCs發(fā)生發(fā)展起作用的轉(zhuǎn)錄因子陸續(xù)被發(fā)現(xiàn)，如Dmrt1對(duì)于Ngn3陽(yáng)性精原細(xì)胞轉(zhuǎn)化為SSCs以恢復(fù)干細(xì)胞池是必需的，并且最新研究表明Dmrt1和Plzf的相互作用，通過(guò)激活Plzf的轉(zhuǎn)錄在穩(wěn)態(tài)環(huán)境下維持SSCs[114,115]。Sohlh1/2能直接結(jié)合到和基因的啟動(dòng)子上調(diào)節(jié)精子發(fā)生，也可以直接結(jié)合到和啟動(dòng)子上抑制并激活的表達(dá)[116,117]。Plzf可以負(fù)向調(diào)控分化相關(guān)基因如和的啟動(dòng)子[118]。過(guò)度表達(dá)PAX7后，與自我更新相關(guān)的基因如和在SSCs中的表達(dá)上調(diào)[119]。近期研究發(fā)現(xiàn)，RHOX10可通過(guò)Dmrt1和Plzf驅(qū)動(dòng)SSCs前體即前精原細(xì)胞(pro-spermatogonia, ProSG)分化[120]。隨著轉(zhuǎn)錄組技術(shù)的發(fā)展，越來(lái)越多的對(duì)SSCs發(fā)生發(fā)展產(chǎn)生影響的轉(zhuǎn)錄因子被發(fā)現(xiàn)。

3.4 參與精子發(fā)生的激素調(diào)控

在SSCs微環(huán)境中除上述的調(diào)控因子外還存在一類(lèi)影響精子發(fā)生的物質(zhì)—激素，其中IGF-1在睪丸的SCs和LCs中表達(dá)，可以促進(jìn)SSCs的增殖、自我更新以及調(diào)節(jié)多能性[43,121]。同時(shí)在體外培養(yǎng)睪丸組織時(shí)，在培養(yǎng)基中添加IGF-1可減少GCs凋亡和促進(jìn)增殖[122]。其具體的促進(jìn)自我更新和增殖的機(jī)制可解釋為IGF-1可能通過(guò)PI3K/AKT信號(hào)轉(zhuǎn)導(dǎo)介導(dǎo)IGF-1/IGF-1R調(diào)控OCT-4等多種轉(zhuǎn)錄因子和GCs多能性，LCs分泌的IGF-1也能以旁分泌的方式促進(jìn)未成熟SCs的增殖[43,123]。促進(jìn)精原細(xì)胞轉(zhuǎn)變?yōu)槌跫?jí)精母細(xì)胞的具體機(jī)制可解釋為下調(diào)導(dǎo)致IGF-1/IGF-1R上調(diào)，級(jí)聯(lián)放大將ERK1/2和PI3K激活[124]。關(guān)于IGF結(jié)合蛋白，PMCs主要分泌IGFBP-2，SCs主要分泌IGFBP-3[125]。除上述因素外，精子發(fā)生也受控于以睪酮、FSH和黃體生成素(luteinizing hormone, LH)為主的外源性控制，LCs和SCs表面分別存在LH和FSH受體，LH可刺激LCs分泌睪酮，睪酮和FSH聯(lián)合作用于SCs，從而影響精子發(fā)生。睪酮等雄激素是正常雄性生殖系統(tǒng)發(fā)育和功能所必需的類(lèi)固醇激素，在維持精子數(shù)目、BTB完整性、減數(shù)分裂的完成等方面起重要作用[39,126]。另外激素可與細(xì)胞因子共同作用，如EGF和FGF2作為細(xì)胞因子培養(yǎng)SSCs形成克隆數(shù)較少，然而當(dāng)添加FSH后SSCs形成的菌落數(shù)量大幅度增加[23]。

4 結(jié)語(yǔ)與展望

SSCs是終身精子發(fā)生的來(lái)源，而SSCs的行為以及命運(yùn)受到微環(huán)境整體因素相互作用的綜合調(diào)控。目前關(guān)于微環(huán)境的研究主要基于單細(xì)胞轉(zhuǎn)錄組測(cè)序技術(shù)(single-cell RNA sequencing, scRNA-seq)，新的技術(shù)正將單細(xì)胞譜分析擴(kuò)展到轉(zhuǎn)錄組之外，如單細(xì)胞空間轉(zhuǎn)錄組、單細(xì)胞翻譯組、單細(xì)胞蛋白質(zhì)組和單細(xì)胞代謝組等技術(shù)，未來(lái)這些策略將會(huì)復(fù)用在一起聯(lián)合分析來(lái)自同一個(gè)細(xì)胞的多種組學(xué)模式，有助于人們定義和發(fā)現(xiàn)豐富的細(xì)胞表型和微環(huán)境因子。目前，對(duì)于SSCs微環(huán)境研究的小鼠模型大多都集中在W/Wv鼠以及白消安處理的小鼠模型等，近年來(lái)也有新的小鼠模型不斷出現(xiàn)，如苯扎氯氨(benzal-konium chloride, BC)特異性去除SCs的細(xì)胞模型等。新的小鼠模型的出現(xiàn)有助于人們對(duì)微環(huán)境的進(jìn)一步研究[127]。對(duì)于GCs的轉(zhuǎn)染技術(shù)也在不斷優(yōu)化，例如綜合考慮轉(zhuǎn)染試劑、質(zhì)粒用量和細(xì)胞數(shù)量等因素優(yōu)化了雞() PGCs轉(zhuǎn)染條件[128]。隨著SSCs微環(huán)境研究的逐漸深入，越來(lái)越多對(duì)精子發(fā)生有影響的因素逐漸被發(fā)現(xiàn)，關(guān)于微環(huán)境的研究也會(huì)越來(lái)越完善。SSCs自身基因的表達(dá)也不可忽視，如近期Wang等[129]發(fā)現(xiàn)在果蠅()睪丸中基因是一個(gè)生殖系統(tǒng)優(yōu)勢(shì)表達(dá)的基因，能影響GCs的自我更新和分化。人類(lèi)SSCs與嚙齒動(dòng)物SSCs共享一些生殖表型但并非完全相同，且人類(lèi)SSCs的長(zhǎng)期培養(yǎng)尚未實(shí)現(xiàn)，大多數(shù)關(guān)于SSCs微環(huán)境的知識(shí)都來(lái)自嚙齒類(lèi)動(dòng)物，關(guān)于人類(lèi)SSCs還需不斷探索。相信在不久的將來(lái)，SSCs微環(huán)境的研究也將為臨床因微環(huán)境缺陷引起的不育癥治療帶來(lái)新的希望，以及開(kāi)發(fā)安全有效的男性避孕方法，同時(shí)對(duì)于體外培養(yǎng)成年SSCs帶來(lái)新思路。此外SSCs在病理和生理?xiàng)l件下的細(xì)胞和分子變化仍有待發(fā)現(xiàn)，這將為SSCs提供有價(jià)值的生物學(xué)信息，還可能揭示雄性不育與癌癥關(guān)聯(lián)的機(jī)制。關(guān)于SSCs微環(huán)境的研究尚存在一些倫理、遺傳以及炎癥等方面的問(wèn)題，相信隨著研究的不斷深入，這些問(wèn)題也終將被解決。今后，SSCs微環(huán)境研究也會(huì)有更加廣闊的應(yīng)用前景。

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Progress on spermatogonial stem cell microenvironment

Zhixin Yu, Pengyu Li, Kai Li, Shiying Miao, Linfang Wang, Wei Song

Spermatogonial stem cells (SSCs) are germ cells (GCs) with long-term self-renewal and differentiation potential in testis, namely tissue stem cells located on the basement membrane, whose self-renewal and differentiation are regulated by the surrounding microenvironment. In recent years, the research of SSCs has made a series of important progress, which brings the hope for the clinical treatment of some male infertility patients. Among them, the microenvironment is particularly important in regulating SSCs. The microenvironment is responsible for integrating the effects of different types of cell components, extracellular matrix, extracellular regulatory molecules and hormones on SSCs, thus regulating the fate of SSCs. The research on SSCs microenvironment has gradually become one of the main contents of stem cell research. In this review, we mainly summarize the cell composition, regulatory factors and characteristics of mouse SSCs microenvironment, thereby providing background information for in-depth study on the structure and function of SSCs microenvironment, and opportunity to find more abundant cell phenotypes and microenvironmental factors through multiple research models in the future.

spermatogonia stem cells; microenvironment; self-renewal; differentiation; hormone regulation

2022-05-05;

2022-07-30;

2022-09-01

國(guó)家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(編號(hào)：2018YFC1003500)和國(guó)家自然科學(xué)基金項(xiàng)目(編號(hào)：31970794，32000586)資助[Supported by the National Key Research and Development Program (No. 2018YFC1003500) and the National Natural Science Foundation of China (Nos. 31970794, 32000586)]

余志鑫，在讀碩士研究生，專(zhuān)業(yè)方向：生物化學(xué)與分子生物學(xué)。E-mail: 15619830345@163.com

宋偉，博士，研究員，研究方向：雄性生殖及相關(guān)重大疾病(不育與腫瘤)的分子機(jī)理與轉(zhuǎn)化應(yīng)用研究。E-mail: songwei@ibms.pumc.edu.cn

10.16288/j.yczz.22-136

(責(zé)任編委: 史慶華)