龐雅平，程愈，郝好杰，劉杰杰，申晶

·基礎(chǔ)研究·

間充質(zhì)干細(xì)胞培養(yǎng)上清輸注對(duì)糖尿病大鼠治療作用的機(jī)制研究

龐雅平，程愈，郝好杰，劉杰杰，申晶

目的 觀察間充質(zhì)干細(xì)胞(MSCs)培養(yǎng)上清輸注對(duì)糖尿病大鼠高血糖紊亂的治療效果，探討MSCs分泌產(chǎn)物促進(jìn)胰島再生的作用機(jī)制。方法 SD大鼠腹腔單次注射大劑量鏈脲佐菌素(STZ，65mg/kg)建立糖尿病大鼠模型，將誘導(dǎo)成功的30只糖尿病大鼠隨機(jī)分為MSCs培養(yǎng)上清治療組(CM，n=15)、培養(yǎng)基治療組(M，n=15)，分別輸注MSCs培養(yǎng)上清、培養(yǎng)基。另將15只正常大鼠作為對(duì)照組。連續(xù)治療3d后每日測血糖，于治療第7天測定血清胰島素及C肽，并行腹腔糖耐量檢查(IPGTT)。對(duì)動(dòng)物胰腺組織標(biāo)本行多重免疫熒光染色，觀察MSCs上清治療后胰島β細(xì)胞再生情況，并進(jìn)一步探究胰島細(xì)胞再生的可能機(jī)制。結(jié)果 與M組比較，治療早期(<7d)CM組糖尿病大鼠血糖降低，血清胰島素及C肽含量明顯增加(P<0.05)。IPGTT結(jié)果顯示，CM組較M組糖尿病大鼠胰島功能明顯增強(qiáng)。胰腺組織多重免疫熒光染色提示，CM組糖尿病大鼠的胰島內(nèi)β細(xì)胞數(shù)量較M組明顯增加(P<0.05)，但仍少于對(duì)照組；β細(xì)胞增殖率較M組及對(duì)照組均顯著提高(P<0.01)。結(jié)論 MSCs分泌產(chǎn)物通過促進(jìn)胰島β細(xì)胞增殖，促進(jìn)胰腺再生，恢復(fù)胰島功能，可用于早期糖尿病的控制與治療。

間質(zhì)干細(xì)胞；胰島；再生；胰島素分泌細(xì)胞

間充質(zhì)干細(xì)胞(MSCs)是一類具有自我復(fù)制和多向分化潛能的成體干細(xì)胞，在多種疾病中參與受損組織的修復(fù)與再生，如心肌梗死[1]、急性腎損傷[2]、創(chuàng)傷[3]等。臨床數(shù)據(jù)顯示[4]，MSCs輸注能降低糖尿病患者的血糖水平，顯著減少外源性胰島素用量。此外，在糖尿病動(dòng)物模型中，MSCs的系統(tǒng)輸注可明顯提高β細(xì)胞質(zhì)量，升高體內(nèi)胰島素水平，有效改善高血糖狀態(tài)[5-6]。盡管MSCs治療為治愈糖尿病帶來了希望，但關(guān)于細(xì)胞治療的安全性尚存在很大爭議，制約了干細(xì)胞治療的推廣應(yīng)用。與此同時(shí)越來越多的學(xué)者發(fā)現(xiàn)，MSCs的分泌效應(yīng)在其促進(jìn)組織損傷再生修復(fù)的過程中發(fā)揮了重要作用[7-10]。本課題組前期研究發(fā)現(xiàn)，輸注MSCs上清在一定程度上可起到類似干細(xì)胞本身輸注的作用，達(dá)到促進(jìn)糖尿病大鼠胰島再生、改善高糖狀態(tài)的效果，但與細(xì)胞本身輸注治療相比在療效上仍存在很大差距，且其促β細(xì)胞再生的具體機(jī)制尚未明確[6]。本實(shí)驗(yàn)旨在對(duì)MSCs上清液進(jìn)行濃縮以增強(qiáng)治療效應(yīng)，同時(shí)對(duì)MSCs分泌產(chǎn)物促進(jìn)胰島β細(xì)胞再生的機(jī)制進(jìn)行初步研究。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物、主要試劑及儀器 8周齡雄性SD大鼠(SPF級(jí))50只，體重200～220g，購自解放軍總醫(yī)院醫(yī)學(xué)實(shí)驗(yàn)動(dòng)物中心(動(dòng)物合格證號(hào)：11400700074794)。L-DMEM、FBS(Hyclone，美國)；KO血清、熒光二抗(Invitrogen，美國)；0.25%胰蛋白酶(Gibco，美國)；Glucagon抗體(1/1500)、Insulin抗體(1/150)、Ki-67(1/30)、H33342核染料(1/2000)、STZ(Sigma，美國)。細(xì)胞培養(yǎng)孵箱(Heraeus Sepatech，德國)；臺(tái)式小型高速離心機(jī)(Eppendorf，德國)；低溫冰凍切片機(jī)(Leica，德國)；激光共聚焦顯微鏡(Olympus，日本)；血糖儀(強(qiáng)生，美國)。

1.2 方法

1.2.1 SD大鼠骨髓間充質(zhì)干細(xì)胞(BM-MSCs)分離、培養(yǎng)及培養(yǎng)上清的獲取 雄性，體重100g的正常SD大鼠麻醉后脫頸處死，用含胎牛血清的LDMEM培養(yǎng)基沖出股骨、脛骨的骨髓接種至培養(yǎng)皿，37℃、5% CO2進(jìn)行原代培養(yǎng)。通過密度梯度離心法和貼壁培養(yǎng)法分離、擴(kuò)增MSCs，待細(xì)胞達(dá)90%以上融合時(shí)使用胰蛋白酶消化傳代。傳至第3代時(shí)更換為KO血清培養(yǎng)基，孵育48h后取培養(yǎng)上清，4℃離心，10倍濃縮待用。

1.2.2 糖尿病模型的制備及分組 35只SD大鼠禁食12h后稱重并經(jīng)尾靜脈采血測基礎(chǔ)血糖，用檸檬酸緩沖液溶解鏈脲佐菌素(STZ)，按照65mg/kg的劑量單次腹腔注射。第3天起每日9:00-10:00尾靜脈采血測血糖，連續(xù)3d血糖值>28mmol/L的大鼠判定為造模成功。將30只成模大鼠隨機(jī)分為MSCs培養(yǎng)上清治療組(CM，n=15)、培養(yǎng)基輸注組(M，n=15)。另將15只正常大鼠作為對(duì)照組。

1.2.3 MSCs培養(yǎng)上清輸注及血清學(xué)指標(biāo)測定 CM組的糖尿病大鼠經(jīng)尾靜脈緩慢注入MSCs培養(yǎng)上清濃縮液，2ml/d，連續(xù)3d，M組輸注等量MSCs培養(yǎng)基。然后每日尾靜脈采血測血糖，于治療第7天尾靜脈采血檢測血清胰島素及C肽水平，并進(jìn)行腹腔糖耐量實(shí)驗(yàn)(IPGTT)檢測：各組大鼠在空腹?fàn)顟B(tài)下經(jīng)腹腔注射葡萄糖(2g/kg)，分別于注射后30、60、90、120min檢測血糖。

1.2.4 胰腺組織標(biāo)本的制備、切片及免疫熒光染色各組大鼠分別于治療后2、7、15、30d經(jīng)戊巴比妥鈉麻醉成功后迅速打開胸腔，PBS經(jīng)主動(dòng)脈快速灌流，再以4%多聚甲醛固定1.5～2.0h，取胰腺組織放入30%PB蔗糖溶液中脫水，至組織沉底后用包埋劑包埋保存于–80℃。取胰腺(中尾部)組織以5μm厚度連續(xù)切片，進(jìn)行insulin/glucagon、insulin/Ki-67免疫熒光染色。激光共聚焦顯微鏡下觀察并采集圖像。

1.3 統(tǒng)計(jì)學(xué)處理 采用SPSS 18.0軟件進(jìn)行分析，所有數(shù)據(jù)以±s表示，多組間比較采用方差分析，進(jìn)一步比較采用LSD-t檢驗(yàn)。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié) 果

2.1 MSCs濃縮上清輸注對(duì)STZ誘導(dǎo)的糖尿病大鼠高血糖的改善作用 CM組糖尿病大鼠在治療早期(<7d)血糖水平明顯降低，從30.64±1.99mmol/L降至20.98±1.46mmol/L。M組的糖尿病大鼠為持續(xù)高糖狀態(tài)，血糖始終維持在30.28±0.75mmol/L，治療前后差異無統(tǒng)計(jì)學(xué)意義。IPGTT結(jié)果顯示，CM組糖尿病大鼠在治療后7d受損胰腺的糖代謝能力明顯改善，且血清胰島素水平及C肽含量較M組明顯增加。但在治療晚期(>7d)大鼠隨機(jī)血糖出現(xiàn)逐漸上升趨勢(shì)，治療后15d血糖升高至治療前水平(圖1)。

2.2 MSCs濃縮上清輸注對(duì)胰島內(nèi)β細(xì)胞數(shù)量的影響 insulin/glucagon免疫熒光染色觀察發(fā)現(xiàn)，STZ注射后大鼠胰腺內(nèi)β細(xì)胞毀損達(dá)95%以上。在治療早期(<7d)，與M組相比，CM組大鼠受損胰島中β細(xì)胞數(shù)量明顯增多，以治療后2d增加最為明顯，增加約3～5倍，β細(xì)胞數(shù)于治療后7d開始逐漸減少，至15d時(shí)與M組比較差異無統(tǒng)計(jì)學(xué)意義(圖2)。

2.3 MSCs濃縮上清輸注對(duì)胰島β細(xì)胞增殖的影響 為明確糖尿病大鼠體內(nèi)新生β細(xì)胞的來源，我們對(duì)胰島細(xì)胞進(jìn)行insulin/Ki-67免疫熒光染色，結(jié)果顯示：在對(duì)照組和M組糖尿病大鼠胰島內(nèi)幾乎觀察不到Ki-67染色陽性的β細(xì)胞，提示成年大鼠胰島內(nèi)β細(xì)胞很少出現(xiàn)增殖，而CM組在治療早期(<7d)胰島β細(xì)胞中Ki-67陽性細(xì)胞比例(4.0%)與對(duì)照組、M組(分別為0.5%、0.14%)相比明顯增加，治療后2d后Ki-67陽性的細(xì)胞數(shù)量最多，但治療晚期(>7d)Ki-67陽性細(xì)胞明顯減少(圖3)。

圖1 MSCs培養(yǎng)上清輸注對(duì)STZ誘導(dǎo)的糖尿病大鼠高血糖的改善作用Fig. 1 MSC supernatant infusion ameliorate the STZ-induced hyperglycemia in DM rats

圖2 MSCs濃縮上清輸注對(duì)胰島內(nèi)β細(xì)胞的數(shù)量的影響(Insulin/glucagon免疫熒光染色)Fig. 2 Effect of infusion of concentrated MSCs supernatant on the number of islet β cells (Insulin/glucagon immunofluorescent staining) Red represents insulin, green represents glucagon; (1)P<0.05, (2)P<0.01

3 討 論

近年來隨著研究深入，MSCs治療糖尿病在臨床試驗(yàn)和動(dòng)物研究中均表現(xiàn)出令人興奮的療效，接受干細(xì)胞治療的糖尿病個(gè)體血糖水平明顯下降，糖耐量異常明顯改善，外源胰島素需要量顯著減少。但細(xì)胞治療的具體作用機(jī)制極為復(fù)雜，回顧文獻(xiàn)，目前已知的機(jī)制有：①免疫調(diào)節(jié)和抗炎作用。MSCs具有特殊的免疫調(diào)節(jié)功能，通過分泌一系列細(xì)胞因子，作用于抗原呈遞細(xì)胞、T細(xì)胞和自然殺傷細(xì)胞等免疫細(xì)胞產(chǎn)生免疫抑制[11]。MSCs還可分泌抗炎因子，參與免疫抑制，進(jìn)而防止炎癥和自身免疫病的發(fā)生[12]。②促進(jìn)胰腺再生。MSCs治療糖尿病的體內(nèi)外實(shí)驗(yàn)提示，MSCs可活化內(nèi)源性干、祖細(xì)胞向胰島素生成細(xì)胞分化[13]，促進(jìn)非β細(xì)胞向β細(xì)胞重編程[14]，促進(jìn)胰島β細(xì)胞增殖[15]，以及抑制細(xì)胞凋亡[7]，進(jìn)而實(shí)現(xiàn)胰腺再生。而其中MSCs分泌的生長因子，如血管內(nèi)皮生長因子(VEGF)[16]、胰島素樣生長因子-1(IGF-1)[17]、肝細(xì)胞生長因子(HGF)[18]等在促進(jìn)胰島內(nèi)血管生成、保護(hù)β細(xì)胞存活、抗凋亡等多種機(jī)制中發(fā)揮重要作用。③改善胰島素抵抗。最新研究發(fā)現(xiàn)，MSCs輸注可有效改善2型糖尿病大鼠周圍靶組織對(duì)胰島素的敏感性[19]。而MSCs分泌的效應(yīng)因子成纖維細(xì)胞生長因子-1(FGF-1)在改善胰島素抵抗中發(fā)揮重要作用[20]。綜上所述，雖然MSCs通過多種途徑促進(jìn)胰腺再生，但究其根本均與其分泌效應(yīng)密切關(guān)聯(lián)。

圖3 MSCs培養(yǎng)上清輸注對(duì)胰島β細(xì)胞增殖的影響(Insulin/Ki-67免疫熒光染色)Fig. 3 Effect of infusion of MSCs supernatant on islet β cell proliferation (Insulin/Ki-67 immunofluorescent staining) Red represents Ki-67, green represents insulin; the arrows indicate the insulin+Ki-67+cells. (1)P<0.01, (2)P<0.01

我們的前期研究證實(shí)，對(duì)糖尿病大鼠進(jìn)行直接MSCs上清輸注在一定程度上確實(shí)可以促進(jìn)糖尿病大鼠的胰島再生，但對(duì)高血糖的改善并不明顯，推測可能與上清液中有效活性分子的濃度太低，上清去細(xì)胞后缺乏持續(xù)的因子分泌有關(guān)。因此，本實(shí)驗(yàn)對(duì)MSCs上清進(jìn)行適度濃縮結(jié)果顯示，MSCs濃縮上清于治療早期可明顯降低糖尿病大鼠的血糖水平，并且給予糖負(fù)荷后可刺激胰島素分泌，而對(duì)照組無明顯變化，表明MSCs濃縮上清治療使受損的胰島功能得到恢復(fù)，盡管這一效應(yīng)僅僅出現(xiàn)于治療早期(<7d)。此外，單次大劑量STZ腹腔注射可導(dǎo)致胰腺β細(xì)胞重度毀損，體內(nèi)β細(xì)胞嚴(yán)重不足，而治療早期胰島內(nèi)β細(xì)胞數(shù)量較對(duì)照組明顯增多，同時(shí)伴隨β細(xì)胞的顯著增殖，其增殖率達(dá)4%～8%，遠(yuǎn)遠(yuǎn)高于健康成年大鼠的β細(xì)胞增殖率(0.5%)[21]。由此推斷MSCs分泌效應(yīng)可能通過提高胰島β細(xì)胞增殖率，從而實(shí)現(xiàn)胰島再生，在治療早期可有效改善高血糖狀態(tài)。

遺憾的是，本研究發(fā)現(xiàn)上述療效僅僅發(fā)生于治療早期，隨著時(shí)間的推移，糖尿病大鼠血糖逐漸升高至治療前的高糖狀態(tài)，且伴隨β細(xì)胞數(shù)量的顯著減少，β細(xì)胞增殖明顯降低。可能原因?yàn)椋孩費(fèi)SCs上清中效應(yīng)因子含量有限，且存在一定的半衰期，僅能維持短期療效，長期效果不顯著；②新生β細(xì)胞無力抵抗高糖環(huán)境的刺激，致新生細(xì)胞凋亡[22]；③持續(xù)的高糖環(huán)境抑制細(xì)胞增殖[23]。

總之，MSCs上清于治療早期可促進(jìn)受損胰島再生，至少部分是通過促進(jìn)β細(xì)胞增殖來實(shí)現(xiàn)的。如何提高M(jìn)SCs上清的療效，延長作用時(shí)間，以及對(duì)發(fā)揮主要療效的細(xì)胞因子的鑒別均有待進(jìn)一步研究。

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Infusion of mesenchymal stem cells culture supernatant ameliorates hyperglycemia disorders in STZ-induced diabetes mellitus rats

PANG Ya-ping1, CHENG Yu2, HAO Hao-jie2, LIU Jie-jie2, SHEN Jing3*1Graduate School of Hebei North University, Zhangjiakou, Hebei 075000, China
2School of Life Science, General Hospital of Chinese PLA, Beijing 100039, China
3Department of Endocrinology, 309 Hospital of Chinese PLA, Beijing 100091, China
*

, E-mail: shenjingshenhua@126.com
This work was supported by the National Natural Science Foundation of China (81200615)

ObjectiveTo evaluate the therapeutic effect of systemic infusion of MSC culture supernatant in STZ-induced diabetic rats, and explore the mechanism of effect of MSC secretion on promoting regeneration of the islet tissues.MethodsThe diabetic animal model was reproduced in 35 SD rats by intraperitoneal injection of a single large dose of streptozotocin (STZ, 65mg/ kg). The 30 successfully induced diabetic rats were randomly divided into MSC culture supernatant infusion group (CM, n=15) and medium infusion group (M, n=15), and in addition, 15 normal rats were used as control. Animals were intravenously transfused with MSC supernatant (CM group) or raw medium (M group), then the contents of blood glucose were determined 3 days after infusion. The serum insulin and C-peptide levels were monitored and the intraperitoneal glucose tolerance test (IPGTT) was performed on the 7th day of infusion to evaluate the therapeutic effect of MSCs supernatant infusion in diabetic rats. Finally, all the experimental animals were sacrificed at indicated time points and the pancreatic tissues were collected for multiple immunofluorescence staining (MIFS), in order to observe the β-cell regeneration after MSCs supernatant infusion, and to further explore the possible mechanism involved in the experiment.ResultsAt the early stage after infusion (<7 days), the blood glucose level declined and the contents of serum insulin and C-peptide increased obviously in CM group as compared with that of M group (P<0.05). IPGTT showed that the islet function was significantly enhanced in CM group compared with M group. MIFS showed that the number of β cells in thedestroyed islets in CM group rats was significantly increased as compared with that of M group rats. In addition, the proliferation rate of β cells was obviously higher in CM group (4%) than in control group (0.5%, P<0.01). ConclusionTreatment with MSCs culture supernatant obviously prompted β cell proliferation in the destroyed islets, resulting in regeneration of the islets with islet function recovery, thus it effectively controls the advance of diabetes.

mesenchymal stem cells; islets of Langerhans; regeneration; insulin-secreting cells

R587.1

A

0577-7402(2015)06-0449-05

10.11855/j.issn.0577-7402.2015.06.05

2015-01-15；

2015-03-23)

(責(zé)任編輯：張小利)

國家自然科學(xué)基金(81200615)

龐雅平，碩士研究生。主要從事間充質(zhì)干細(xì)胞與糖尿病治療方面的研究

075000 河北張家口 河北北方學(xué)院研究生院(龐雅平)；100039 北京 解放軍總醫(yī)院生命科學(xué)院(程愈、郝好杰、劉杰杰)；100091 北京 解放軍309醫(yī)院內(nèi)分泌科(申晶)

申晶，E-mail：shenjingshenhua@126.com