基于經(jīng)血源性子宮內(nèi)膜干細胞的中樞神經(jīng)系統(tǒng)疾病治療的研究進展

劉蒙蒙，成欣然，李凱凱，許鳴銳，吳永繼，王夢麗，張倩如，閆文勇，羅暢，趙善廷

1 西北農(nóng)林科技大學(xué) 動物醫(yī)學(xué)院 神經(jīng)生物學(xué)實驗室，陜西 楊凌 712100

2 西北農(nóng)林科技大學(xué) 食品科學(xué)與工程學(xué)院，陜西 楊凌 712100

3 西北農(nóng)林科技大學(xué) 創(chuàng)新實驗學(xué)院，陜西 楊凌 712100

與外周神經(jīng)系統(tǒng)損傷修復(fù)相比，中樞神經(jīng)系統(tǒng)疾病的治療是公認的世界難題，多項研究表明干細胞治療有望解決此題。經(jīng)血源性子宮內(nèi)膜干細胞 (Menstrual blood-derived stem cells，MenSCs)是新近發(fā)現(xiàn)的干細胞，既具有來源廣、取材易、多向分化潛能等特性，又具有無倫理學(xué)問題、可實現(xiàn)個性化治療及免疫原性低等臨床優(yōu)勢。因此，結(jié)合本實驗室之前對于中樞神經(jīng)系統(tǒng)疾病原理的探究以及對 MenSCs研究的初步結(jié)果，本文主要就MenSCs的特性及其在中風(fēng)、脊髓損傷修復(fù)、惡性膠質(zhì)瘤和多發(fā)性硬化等中樞神經(jīng)系統(tǒng)疾病治療中的應(yīng)用作一綜述，并展望了 MenSCs的應(yīng)用前景。

1 經(jīng)血源性子宮內(nèi)膜干細胞

1.1 經(jīng)血源性子宮內(nèi)膜干細胞的發(fā)現(xiàn)及來源

因血管生成是月經(jīng)周期中子宮內(nèi)膜增生的關(guān)鍵環(huán)節(jié)，Meng等[1]提出能在月經(jīng)血中分離出干細胞這一假說且得到證實，并將此干細胞命名為子宮內(nèi)膜再生細胞。之后，Patel等[2]提取、分離、命名該細胞為經(jīng)血源性子宮內(nèi)膜基質(zhì)干細胞，并證明該細胞能分化為中、外兩個胚層細胞系。經(jīng)研究發(fā)現(xiàn)MenSCs的確切來源是月經(jīng)期間子宮內(nèi)膜功能層脫落而來的月經(jīng)血，而不是源于骨髓間充質(zhì)干細胞[3]。子宮壁由內(nèi)向外分為3層，即子宮內(nèi)膜、肌層和漿膜，而子宮內(nèi)膜又分為基底層和功能層。月經(jīng)周期分為3個時期：增殖期、分泌期和月經(jīng)期。在月經(jīng)期間，子宮內(nèi)膜只有功能層脫落。月經(jīng)周期受到性類固醇激素等的調(diào)控[4](圖1)，在雌激素等調(diào)控的增殖期，子宮內(nèi)膜在10 d內(nèi)增厚5?7 mm[5]；在黃體酮等調(diào)控的分泌期，腺體和基質(zhì)成熟[6]；在分泌晚期，黃體退化，引起雌激素和黃體酮的分泌降低，觸發(fā)子宮內(nèi)膜功能層脫落[7]。因而，女性月經(jīng)期間子宮內(nèi)膜功能層脫落的月經(jīng)血可以提取并分離出MenSCs。

圖1 在月經(jīng)周期中人子宮內(nèi)膜功能層的變化[4]Fig. 1 Schematic of changes in the human endometrium during the menstrual cycle, illustrating the growth,differentiation and shedding of the functionalis layer[4].

1.2 MenSCs的分離、提取及鑒定

方法一是通過密度梯度離心分離出單個核細胞并成功誘導(dǎo)其分化為心肌細胞、呼吸上皮細胞、神經(jīng)細胞、肌細胞、內(nèi)皮細胞、胰腺細胞、脂肪細胞和成骨細胞；經(jīng)流式細胞儀分析該細胞標(biāo)志物并得到其表型特征為 Oct-4、CD105、CD44、CD73和 CD9等陽性，而 Nanog、SSEA-4、CD45、CD34和CD14等陰性[1]。這是首次證明從月經(jīng)血中可以分離出干細胞且該細胞具有分化為內(nèi)、中、外3個胚層細胞系的潛能。方法二是直接離心取沉淀培養(yǎng)，用與子宮內(nèi)膜高增殖性密切相關(guān)的CD117 (c-kit) 微磁珠技術(shù)篩選細胞，獲得的細胞經(jīng)分析陽性表達SSEA-4以及Oct-4，且可誘導(dǎo)分化為軟骨細胞、脂肪細胞、成骨細胞、心肌細胞和神經(jīng)細胞，證明該細胞能分化為中、外兩個胚層細胞系[2]。

不同的收集和處理方法得到的 MenSCs可能會在月經(jīng)血中提取出具有不同生物學(xué)特性的細胞群[1-2,8]。不同的研究者在 MenSCs是否表達表面標(biāo)志物SSEA-4方面的結(jié)果仍有爭議[1-2]。而研究表明 MenSCs表達端粒酶催化亞基和 Nanog[9]，這對于體外具有高增殖活性的干細胞來說是常見的。此外，MenSCs不表達HLA-DR分子和低表達HLAⅠ類分子[10]，這些都進一步為MenSCs的臨床應(yīng)用奠定了基礎(chǔ)。

1.3 MenSCs的體外分化

最初，Meng等[1]用含 GlutaMax和 hFGA-4的神經(jīng)誘導(dǎo)培養(yǎng)基誘導(dǎo) MenSCs分化為表達星形膠質(zhì)細胞標(biāo)記物 GFAP和神經(jīng)干細胞標(biāo)記物Nestin的神經(jīng)樣細胞 (Neural like cells，NLCs)。緊接著 Patel等[2]在誘導(dǎo)培養(yǎng)基中添加了 N2、bFGF以及EGF等誘導(dǎo)MenSCs分化為少突膠質(zhì)樣細胞、神經(jīng)元樣細胞以及神經(jīng)祖細胞樣細胞。后來，Zemel’ko等[9]證明相比于骨髓間充質(zhì)干細胞和脂肪干細胞，全反式維甲酸對 MenSCs的神經(jīng)分化是必需的。這些方法都為后來的研究奠定了重要基礎(chǔ)。總而言之，MenSCs在體外誘導(dǎo)分化為NLCs與神經(jīng)營養(yǎng)因子或其類似物緊密相連，在誘導(dǎo)條件下，一般先轉(zhuǎn)變?yōu)樯窠?jīng)祖細胞樣細胞，再最終分化為其他NLCs。大量MenSCs體外分化機制的探究為許多不治之癥帶來了希望。

2 MenSCs與中樞神經(jīng)系統(tǒng)疾病的治療

腦部疾病是引起人類死亡的第三大原因，而干細胞可以適時地調(diào)節(jié)炎癥，消除細胞死亡，保留神經(jīng)功能。之前的許多研究中，已經(jīng)成功地將MenSCs分化成各種細胞系，包括膠質(zhì)樣細胞[11]。與骨髓間充質(zhì)干細胞相比 (表 1)，MenSCs具有長期的自我更新能力和較強的增殖能力，較小的核型異常風(fēng)險，不會引發(fā)免疫原性反應(yīng)或腫瘤形成[12-13]。因此，MenSCs更有可能成為治療中樞神經(jīng)系統(tǒng)疾病的“潛力股”。另外，MenSCs取自“廢物”——月經(jīng)血，避免了胚胎干細胞的倫理問題以及骨髓干細胞取材的侵入性，這些特性使得 MenSCs的臨床應(yīng)用會變得更加廣泛。因此，我們對 MenSCs在中樞神經(jīng)系統(tǒng)疾病如中風(fēng)[19,24-25]、脊髓損傷[26]、惡性膠質(zhì)瘤[27]和多發(fā)性硬化[28]等疾病治療中的應(yīng)用(表2) 作一闡述。

表1 經(jīng)血源性子宮內(nèi)膜干細胞與骨髓間充質(zhì)干細胞的比較Table 1 Comparison of menstrual blood-derived endometrial stem cells (MenSCs) with bone marrow mesenchymal stem cells (BM-MSCs)

表2 經(jīng)血源性子宮內(nèi)膜干細胞在中樞神經(jīng)系統(tǒng)疾病治療中的應(yīng)用Table 2 Application of menstrual blood-derived endometrial stem cells in the treatment of central nervous system diseases

2.1 MenSCs與中風(fēng)

“腦卒中”又稱“中風(fēng)”、“腦血管意外”，是一種急性腦血管疾病，具有發(fā)病率高、死亡率高和致殘率高的特點，也是導(dǎo)致中國成年人殘疾的首要原因，然而一直缺乏有效的治療手段，干細胞治療有望解決此難題。

眾所周知，中風(fēng)不僅僅影響神經(jīng)元的功能，而且涉及到與其周圍免疫系統(tǒng)相互作用的“血管神經(jīng)纖維瘤”中的腦細胞及其細胞外基質(zhì)。由于這些原因，中風(fēng)的治療應(yīng)針對這些系統(tǒng)，而不是只針對個別的損傷過程，才能避免過去臨床轉(zhuǎn)化開發(fā)特定神經(jīng)保護藥物的失敗嘗試。Borlongan等[29]使用Patel等[2]描述的MenSCs研究中風(fēng)的體外和體內(nèi)模型的治療。研究發(fā)現(xiàn)這些細胞提供了一些保護原代神經(jīng)元免受氧-葡萄糖剝奪的物質(zhì)。進一步研究發(fā)現(xiàn)這些物質(zhì)能夠發(fā)揮類似的神經(jīng)保護作用，這可能與血管內(nèi)皮生長因子、腦源性生長因子和神經(jīng)營養(yǎng)因子 3有關(guān)。早先，關(guān)于其他干細胞系的研究也發(fā)現(xiàn)了一種或多種因子的釋放[30-31]及其對卒中治療的潛在益處[32]，該研究明確表示無論是在腦內(nèi)還是在靜脈內(nèi)自體移植 MenSCs均未顯示免疫抑制；實驗誘導(dǎo)成年大鼠缺血性卒中后，其行為學(xué)和組織學(xué)損傷顯著降低。因此，移植MenSCs而得的神經(jīng)結(jié)構(gòu)和血管生成能力可以成為中風(fēng)治療的有效途徑，也支持它們成為用于其他基底神經(jīng)節(jié)疾病，如帕金森病和亨廷頓病治療的干細胞來源。

2.2 MenSCs與脊髓損傷修復(fù)

脊髓損傷 (Spinal cord injury，SCI) 是一種創(chuàng)傷性損傷，可導(dǎo)致運動神經(jīng)元或感覺神經(jīng)元的損失。與周圍神經(jīng)系統(tǒng)相比，脊髓的有限再生能力是源于創(chuàng)傷后環(huán)境，如缺血、炎癥、免疫應(yīng)答和膠質(zhì)瘢痕的形成[33]。已經(jīng)證明，干細胞治療可通過釋放一系列內(nèi)源性修復(fù)的營養(yǎng)因子或通過分化成神經(jīng)元或神經(jīng)膠質(zhì)細胞以替換受損細胞來促進SCI后的神經(jīng)元再生[34-36]。另外，三維支架通過模擬內(nèi)源性微環(huán)境更有利地支持細胞存活、增殖和體內(nèi)分化。故將工程支架與干細胞結(jié)合可能成為治療SCI的有前途的策略。

MenSCs作為具有多種臨床應(yīng)用優(yōu)勢的干細胞，已有研究將其種植在一種納米纖維上，再植入到背側(cè)半透明的大鼠模型中，可以減輕二次反應(yīng)，促進神經(jīng)元再生、軸突髓鞘再生和運動功能恢復(fù)[26]。此外，作為抗炎和神經(jīng)保護介質(zhì)的番茄紅素的同時，給藥可以抑制神經(jīng)變性過程并幫助改善神經(jīng)元再生。這表明MenSCs的移植并結(jié)合支架可以更好地促進損傷部位細胞的恢復(fù)，成為有效治療SCI的方法。

2.3 MenSCs與膠質(zhì)瘤

神經(jīng)膠質(zhì)瘤是中樞神經(jīng)系統(tǒng)中最常見和最惡性的腦腫瘤。腫瘤細胞的侵襲性和浸潤性以及有效治療的困難導(dǎo)致預(yù)后很差。盡管手術(shù)、放療、化療甚至基因治療已被廣泛應(yīng)用，但膠質(zhì)瘤的5年生存率仍低于10%[37]。因此，迫切需要一種消除侵襲性腫瘤細胞而不損傷正常腦組織的新的治療手段。

研究表明，腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體(Tumor necrosis factor-related apoptosis-inducing ligand，TRAIL) 可以通過激活凋亡途徑誘導(dǎo)癌細胞凋亡。結(jié)構(gòu)分析表明，分泌型TRAIL (sTRAIL)含有蛋白質(zhì)的受體結(jié)合域，可用于選擇性觸發(fā)癌細胞凋亡而不損害正常細胞[38]。而Wang等[27]結(jié)合sTRAIL與MenSCs靶向治療惡性膠質(zhì)瘤，為神經(jīng)膠質(zhì)瘤的治療帶來了新的思路。在該研究中，當(dāng)用作治療藥物的基因遞送載體時，MenSCs顯示人類惡性膠質(zhì)瘤的向性。而感染過表達sTRAIL的有效腺病毒血清型35載體的MenSCs在體外和體內(nèi)均顯示出顯著的抗腫瘤作用。在此，MenSCs既具有干細胞增殖、分化的優(yōu)點，又作為sTRAIL等藥物靶向治療的載體，正是治療膠質(zhì)瘤的希望所在。

2.4 MenSCs與多發(fā)性硬化

多發(fā)性硬化癥 (Multiple sclerosis, MS) 是一種常見的中樞神經(jīng)脫髓鞘疾病，伴隨著膠質(zhì)纖維增生而形成鈣化斑，多見于視神經(jīng)、脊髓和腦干。

目前，雖然已有多種疾病緩解療法 (Diseasemodifying therapies，DMTs) 獲得批準(zhǔn)治療MS[39]，但仍沒有有效藥物能阻止該疾病進展或直接促進已有中樞神經(jīng)系統(tǒng)損傷的修復(fù)。使用干細胞治療不僅可以減弱免疫反應(yīng)，還能更好地促進內(nèi)源性修復(fù)機制的運行。Zhong等[28]認為MenSCs是具有多能分化活性和誘導(dǎo)新血管發(fā)生能力的間充質(zhì)干細胞群，在體外和動物體內(nèi)的研究都表明MenSCs具有免疫特性，在某些情況下能主動抑制免疫應(yīng)答，并報道了 1例靜脈內(nèi)和鞘內(nèi)注射MenSCs以及3例鞘內(nèi)注射MenSCs臨床治療多發(fā)性硬化癥患者的初步安全性研究。經(jīng)最長超過一年的隨訪，病人并沒有出現(xiàn)免疫反應(yīng)或治療相關(guān)的不良反應(yīng)。這些初步數(shù)據(jù)表明 MenSCs在臨床治療的可行性，并支持使用這種新型干細胞治療疾病的進一步研究。

Naddafi等[40]研究表明尼古丁在多發(fā)性硬化癥實驗?zāi)Ｐ椭芯哂斜Ｗo作用。而后，Mahfouz等[41]揭示間充質(zhì)干細胞和尼古丁的組合可以進一步改善MS，可成為MS治療的有希望的策略。所以，以細胞為基礎(chǔ)的治療 (移植、動員和藥物治療) 可能有助于退行性機制導(dǎo)致的多發(fā)性硬化癥的治療，但這一假設(shè)有待進一步證實[42]。因此，MenSCs這種無入侵性、來源廣、免疫原性較低的干細胞結(jié)合藥物一起治療MS或許是一種不錯的選擇。

3 展望

移植的干細胞可以提供新的神經(jīng)元以及分泌細胞因子以形成新的功能性神經(jīng)回路并促進軸突再生[43-45]。基于干細胞的治療方法可以促進神經(jīng)元再建、抑制細胞凋亡、軸突再生和髓鞘形成。最新研究報道，神經(jīng)干細胞和間充質(zhì)干細胞已經(jīng)用于損傷脊髓的細胞替代療法。然而，神經(jīng)干細胞治療脊髓損傷的結(jié)果并不理想，很大程度上是由于神經(jīng)膠質(zhì)瘢痕組成以及神經(jīng)干細胞體內(nèi)分化的復(fù)雜性而導(dǎo)致的突觸靶向再生的阻礙[46-48]。此外，神經(jīng)干細胞治療還存在以下問題：1) 移植后的神經(jīng)干細胞存活時間短[49]；2) 所移植的神經(jīng)干細胞純度及其可能與其他宿主神經(jīng)細胞之間產(chǎn)生的不可預(yù)測的相互作用[49-50]；3) 移植后的神經(jīng)干細胞可能逃脫分化和選擇過程并在移植部位擴大形成腫瘤[50-51]。而間充質(zhì)干細胞表現(xiàn)出的下調(diào)促凋亡因子、上調(diào)抗細胞凋亡分子以及阻礙軸突脫髓鞘和退化的能力，已被提倡作為干細胞治療的“明日之星”[52]。但是，宿主免疫應(yīng)答、神經(jīng)分化的缺乏和移植細胞的低存活率仍是使用間充質(zhì)干細胞的限制[53]。

此時，“應(yīng)召而來”的 MenSCs是適用于細胞治療的新型的干細胞來源[54-55]。這些細胞不僅具有分離、提取的非侵入性和快速擴增特性，還表現(xiàn)出高增殖潛力，有分化成為具神經(jīng)源性細胞類型的能力[2,56]。研究表明，MenSCs免疫原性較低，已經(jīng)在治療子宮內(nèi)膜異位癥[57]、卵巢早衰癥[58]、Asherman綜合癥[59]等子宮疾病、高血糖癥[60]、膠原誘導(dǎo)的關(guān)節(jié)炎和異物移植物抗宿主病[10]、急性肝功能衰竭[61]、皮膚創(chuàng)傷[62]以及急性肺損傷[63]等疾病的研究中得以證實。

值得一提的是，間充質(zhì)干細胞的免疫調(diào)節(jié)機制并不總是相同，而是取決于眾多相關(guān)因素的相互作用，需要更進一步的研究來闡述其在不同疾病狀態(tài)下的特異效應(yīng)[64]，這對于MenSCs來說也不例外。此外，干細胞包括 MenSCs的治療仍面臨著很多問題，如移植后細胞的存活、細胞命運、維持指定的細胞分化表型、避免畸胎瘤的形成及移植的安全性等問題。為解決這些問題，將組織工程與干細胞技術(shù)結(jié)合應(yīng)用可能是一個非常不錯的選擇，如上述膠質(zhì)瘤和脊髓損傷的治療。因此MenSCs在臨床應(yīng)用之前不僅需要有充足的研究積淀，還需要進行全面可靠的安全評估。

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