許聿新，井慶平，趙翠紅(淄博市第一醫(yī)院，山東淄博255200)

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·論著·

骨髓間充質(zhì)干細(xì)胞旁分泌效應(yīng)對胰腺β細(xì)胞增殖、凋亡及分泌功能的影響

許聿新，井慶平，趙翠紅(淄博市第一醫(yī)院，山東淄博255200)

摘要：目的觀察骨髓間充質(zhì)干細(xì)胞(BMSCs)旁分泌效應(yīng)對胰腺β細(xì)胞系INS-1細(xì)胞增殖、凋亡及胰島素分泌的影響。方法培養(yǎng)BMSCs，以胰腺提取物(RPE)模擬胰腺微環(huán)境培養(yǎng)BMSCs后，獲取BMSCs的條件培養(yǎng)液(BMSCs-CM)。將細(xì)胞分為A組、B組、C組，分別以BMSCs-CM、不含RPE的條件培養(yǎng)液、等量RPMI 1640培養(yǎng)液培養(yǎng)。采用MTT法觀察各組細(xì)胞增殖情況，計(jì)算細(xì)胞增殖率。采用AnnexinⅤ/PI法觀察各組細(xì)胞凋亡情況，計(jì)算細(xì)胞凋亡率。應(yīng)用放射免疫法(RIA)測定各組胰島素水平。結(jié)果 A組細(xì)胞增殖率高于B組及C組(P均<0.05)。A組細(xì)胞凋亡率低于B組及C組(P均<0.05)。在葡萄糖溶液終濃度為5.6及20.0 mmol/L時(shí)，A組胰島素水平均高于B組及C組(P均<0.05)。結(jié)論BMSCs-CM培養(yǎng)INS-1細(xì)胞后可促進(jìn)其增殖、抑制其凋亡并促進(jìn)其分泌胰島素，BMSCs可能通過旁分泌效應(yīng)發(fā)揮以上作用。

關(guān)鍵詞：干細(xì)胞，骨髓；胰腺β細(xì)胞；細(xì)胞因子；凋亡；增殖

糖尿病的主要發(fā)病機(jī)制之一為胰島β細(xì)胞的增殖及凋亡失衡，導(dǎo)致胰島β細(xì)胞團(tuán)不足，不能分泌充足的胰島素。骨髓間充質(zhì)干細(xì)胞(BMSCs)是重要的種子細(xì)胞，移植BMSCs到胰腺后可以改善糖尿病大鼠的高血糖狀態(tài)，但其機(jī)制尚未明確[1,2]。研究發(fā)現(xiàn)，移植到體內(nèi)的BMSCs并不能直接分化為胰島素分泌細(xì)胞，從而增殖為有功能的胰島β細(xì)胞團(tuán)[3]。但BMSCs在不同的微環(huán)境影響下可以分泌不同的細(xì)胞因子，從而調(diào)節(jié)周圍細(xì)胞的增殖、凋亡及血管生成等，發(fā)揮重要的旁分泌作用。因此，BMSCs旁分泌效應(yīng)可能在改善靶器官功能、抗凋亡方面發(fā)揮重要的作用。我們前期研究發(fā)現(xiàn)，以胰腺提取物(RPE)模擬胰腺微環(huán)境培養(yǎng)BMSCs后，可使BMSCs通過旁分泌效應(yīng)分泌多種對胰腺具有保護(hù)作用的細(xì)胞因子[4]。但這種BMSCs旁分泌效應(yīng)對胰腺β細(xì)胞增殖、凋亡及胰島素分泌的影響尚不明確。 2015 年1～6月，我們用RPE處理BMSCs后的條件培養(yǎng)液(BMSCs-CM)孵育胰腺β細(xì)胞系胰島細(xì)胞瘤INS-1細(xì)胞，觀察BMSCs旁分泌效應(yīng)對INS-1細(xì)胞增殖、凋亡及胰島素分泌的影響，探討B(tài)MSCs移植治療糖尿病的機(jī)制。

1材料與方法

1.1動物及試劑雄性Wistar大鼠20只，體質(zhì)量160～220 g，6～8周齡，清潔級，購自山東大學(xué)實(shí)驗(yàn)動物中心。大鼠INS-1細(xì)胞由佛羅里達(dá)大學(xué)醫(yī)學(xué)院饋贈。主要試劑：RPE參照文獻(xiàn)[4]制備，包含胰腺中多種多肽及細(xì)胞因子；FITC-羊抗鼠二抗IgG(Beckman Coulter公司)；PI(美國Sigma公司)；AnnexinⅤ-FITC(美國BD公司)。

1.2BMSCs培養(yǎng)及鑒定處死大鼠，采用全骨髓貼壁培養(yǎng)法分離培養(yǎng)BMSCs。48 h后，可見少量貼壁細(xì)胞，經(jīng)過3代后BMSCs基本達(dá)到形態(tài)均一。流式細(xì)胞儀分析第3代BMSCs細(xì)胞表型，以細(xì)胞高表達(dá)CD44和CD90，而CD34、CD45低表達(dá)或表達(dá)陰性，為BMSCs培養(yǎng)成功。

1.3BMSCs-CM制備[5,6]取第3代BMSCs，培養(yǎng)于含10%FBS的LG-DMEM的培養(yǎng)板中，培養(yǎng)基中加入RPE培養(yǎng)7 d，然后用無血清培養(yǎng)液繼續(xù)培養(yǎng)48 h，獲得BMSCs-CM。

1.4分組及干預(yù)方法將INS-1細(xì)胞分為A組、B組、C組，分別以BMSCs-CM、不含RPE的條件培養(yǎng)液、等量RPMI 1640培養(yǎng)液培養(yǎng)。

1.5BMSCs-CM對INS-1細(xì)胞增殖的影響觀察采用MTT法。INS-1細(xì)胞接種于96孔板，每孔100 μL，5×103個(gè)/孔，培養(yǎng)24 h。各組處理同1.4。每組設(shè)5個(gè)復(fù)孔，培養(yǎng)44 h。吸去上清，加入新鮮RPMI 1640培養(yǎng)液80 μL，再加入MTT溶液，繼續(xù)培養(yǎng)4 h。每孔加入二甲基亞砜溶解產(chǎn)物150 μL。在酶聯(lián)免疫檢測儀490 nm處測量各孔的吸光度值(OD)，每組設(shè)定6復(fù)孔。細(xì)胞增殖率=(實(shí)驗(yàn)組平均OD值-對照孔平均OD值)/對照孔平均OD值×100%。

1.6BMSCs-CM對INS-1細(xì)胞凋亡的影響觀察采用AnnexinⅤ/PI法。INS-1細(xì)胞培養(yǎng)于含10%FBS的RPMI 1640培養(yǎng)液的24孔板，約5×105/孔，培養(yǎng)24 h后，改為無血清的RPMI 1640培養(yǎng)液。各組處理同1.4。孵育24 h后，添加促凋亡細(xì)胞因子混合物(IL-1β 10 ng/mL、TNF-α 50 ng/mL及 IFN-γ 50 ng/mL)孵育18 h。收集細(xì)胞，重懸于195 μL結(jié)合液。加入Annexin V-FITC 5 μL，室溫避光孵育10 min。加入結(jié)合液重懸細(xì)胞，碘化丙啶染色。熒光顯微鏡觀察凋亡細(xì)胞，正常INS-1細(xì)胞不被熒光標(biāo)記，早期凋亡細(xì)胞被Annexin V染色，鏡下呈綠色；晚期凋亡細(xì)胞或壞死細(xì)胞被PI染色或同時(shí)被Annexin V及PI染色，鏡下見細(xì)胞核呈紅色，或細(xì)胞核的紅色和細(xì)胞膜的綠色同時(shí)存在。隨機(jī)選取6個(gè)視野，流式細(xì)胞儀計(jì)數(shù)早期凋亡細(xì)胞數(shù)、晚期凋亡數(shù)及凋亡細(xì)胞總數(shù)。細(xì)胞凋亡率=凋亡細(xì)胞總數(shù)/細(xì)胞總數(shù)×100%。

1.7BMSCs-CM對INS-1細(xì)胞胰島素分泌的影響觀察INS-1細(xì)胞培養(yǎng)于24孔板中，調(diào)整細(xì)胞數(shù)為5×104/L，孵育48 h。各組處理同1.4。溫箱孵育48 h。每孔中加入葡萄糖溶液，調(diào)整終濃度分別為5.6 mmol/L及20.0 mmol/L，孵育4 h，取樣500 μL，應(yīng)用放射免疫法(RIA)測定胰島素濃度。

2結(jié)果

2.1BMSCs-CM對INS-1細(xì)胞增殖的影響A組細(xì)胞增殖率高于B組及C組(P均<0.05)。B組與C組細(xì)胞增殖率差異無統(tǒng)計(jì)學(xué)意義(P>0.05)。見表1。

2.2BMSCs-CM對INS-1細(xì)胞凋亡的影響 A組細(xì)胞凋亡率低于B組及C組(P均<0.05)。三組早期及晚期凋亡細(xì)胞數(shù)差異均無統(tǒng)計(jì)學(xué)意義(P均>0.05)。見表1。

2.3BMSCs-CM對INS-1細(xì)胞胰島素分泌的影響在葡萄糖溶液終濃度為5.6 mmol/L及20.0 mmol/L時(shí)，A組胰島素水平高于B組及C組(P均<0.05)。見表1。

表1　三組細(xì)胞增殖率、凋亡率、凋亡細(xì)胞數(shù)及胰島素水平比較±s)

注：與A組比較，*P<0.05。

3討論

BMSCs在不同微環(huán)境下的調(diào)控下，可以分泌不同的細(xì)胞因子，這種效應(yīng)被稱為BMSCs旁分泌效應(yīng)[7]。BMSCs分泌的多種細(xì)胞因子可通過作用于鄰近細(xì)胞而發(fā)揮旁分泌作用，廣泛參與細(xì)胞增殖、凋亡、免疫調(diào)節(jié)、血管再生等病理生理過程[8]。研究發(fā)現(xiàn)，低氧環(huán)境培養(yǎng)BMSCs后，培養(yǎng)液中多種具有心肌保護(hù)作用的細(xì)胞因子表達(dá)上調(diào)，表明BMSCs旁分泌效應(yīng)可能發(fā)揮抑制心肌細(xì)胞的凋亡，促進(jìn)其增殖的作用[9,10]。應(yīng)用模擬胰腺微環(huán)境的RPE預(yù)處理后，BMSCs可以顯著增加具有胰腺保護(hù)作用的細(xì)胞因子的分泌，也表明BMSCs旁分泌效應(yīng)可能發(fā)揮修復(fù)胰腺的作用。由于BMSCs移植到糖尿病模型體內(nèi)并不能分化為胰島素分泌細(xì)胞、替代受損的胰島細(xì)胞，因此考慮高血糖的緩解可能與BMSCs移植后通過旁分泌效應(yīng)分泌多種降血糖細(xì)胞因子有關(guān)，進(jìn)而促進(jìn)內(nèi)源性胰島細(xì)胞增殖、抑制其凋亡。

胰島β細(xì)胞數(shù)量的維持是β細(xì)胞增殖及凋亡的動態(tài)平衡。正常成年胰腺β細(xì)胞被認(rèn)為是相對分化成熟的細(xì)胞，但被細(xì)胞因子激活后可出現(xiàn)增殖，這些細(xì)胞因子主要有胰島素樣生長因子(IGF-1)、肝細(xì)胞生長因子(HGF)、堿性成纖維細(xì)胞生長因子(bFGF)、VEGF等[11]。它們與細(xì)胞表面的受體相結(jié)合后，可發(fā)揮促進(jìn)有絲分裂、促進(jìn)細(xì)胞增殖、抑制細(xì)胞凋亡、修復(fù)損傷的作用。研究發(fā)現(xiàn)，應(yīng)用IGF-1處理胰島β細(xì)胞可以抑制細(xì)胞因子誘導(dǎo)的胰島β細(xì)胞凋亡，減少NO的合成，促進(jìn)胰島細(xì)胞分泌胰島素[12]。HGF可通過對抗氧化應(yīng)激來減少游離脂肪酸誘導(dǎo)的RINm5F凋亡，對胰島細(xì)胞具有促進(jìn)增殖的作用[13]。bFGF及VEGF不僅對胰腺細(xì)胞具有促增殖作用，還可通過促進(jìn)血管生成改善胰腺血供、促進(jìn)胰腺修復(fù)[14,15]。本研究發(fā)現(xiàn)， A組增殖率高于B組及C組，提示應(yīng)用REP制備的BMSCs-CM可促進(jìn)INS-1細(xì)胞的增殖；A組細(xì)胞凋亡率低于B組及C組，提示BMSCs-CM可抑制IL-1β、TNF-α和IFN-γ誘導(dǎo)的INS-1細(xì)胞的凋亡；在葡萄糖溶液終濃度為5.6 mmol/L及20.0 mmol/L時(shí)，A組胰島素水平均高于B組及C組，提示BMSCs-CM可促進(jìn)INS-1細(xì)胞分泌胰島素的能力，考慮以上效應(yīng)均與BMSCs通過旁分泌效應(yīng)分泌具有胰腺保護(hù)作用的細(xì)胞因子有關(guān)。

綜上所述，BMSCs-CM培養(yǎng)INS-1細(xì)胞后可促進(jìn)INS-1細(xì)胞的增殖、抑制其凋亡并促進(jìn)其分泌胰島素，BMSCs可能通過分泌多種胰腺保護(hù)細(xì)胞因子的旁分泌效應(yīng)發(fā)揮以上作用，可能是移植BMSCs后逆轉(zhuǎn)高血糖狀態(tài)的重要機(jī)制。

參考文獻(xiàn)：

[1] Aali E, Mirzamohammadi S, Ghaznavi H, et al. A comparative study of mesenchymal stem cell transplantation with its paracrine effect on control of hyperglycemia in type 1 diabetic rats[J]. J Diabetes Metab Disord, 2014,13(1):76.

[2] Gunasekaran U, Gannon M. Type 2 diabetes and the aging pancreatic beta cell[J]. Aging (Albany NY), 2011,3(6):565-575.

[3] Bell GI, Meschino MT, Hughes-Large JM, et al. Combinatorial human progenitor cell transplantation optimizes islet regeneration through secretion of paracrine factors[J]. Stem Cells Dev, 2012,21(11):1863-1876.

[4] 許聿新,井慶平.不同胰腺提取物對大鼠骨髓間充質(zhì)干細(xì)胞分泌細(xì)胞因子的影響[J]. 山東醫(yī)藥,2015, 55(1):25-27.

[5] Benariba N, Bellakdhar W, Djaziri R, et al. Protective action of seed extracts against the deleterious effect of streptozotocin on both glucose-stimulated insulin release from rat pancreatic islets and glucose homeostasis[J]. Biomed Rep, 2013,1(1):119-121.

[6] Lee J, Han DJ, Kim SC. In vitro differentiation of human adipose tissue-derived stem cells into cells with pancreatic phenotype by regenerating pancreas extract[J]. Biochem Biophys Res Commun, 2008,375(4):547-551.

[7] Kono TM, Sims EK, Moss DR, et al. Human adipose-derived stromal/stem cells protect against STZ-induced hyperglycemia: analysis of hASC-derived paracrine effectors[J]. Stem Cells, 2014, 32(7):1831-1842.

[8] 楊媛媛, 周諾. 骨髓間充質(zhì)干細(xì)胞作為組織工程種子細(xì)胞的研究進(jìn)展[J]. 廣西醫(yī)學(xué), 2010,25(5): 586-589.

[9] Sanina C, Hare JM, Mesenchymal stem cells as a biological drug for heart disease: where are we with cardiac cell-based therapy[J]. Circ Res, 2015,117(3): 229-233.

[10] Gnecchi M, Danieli P, Cervio E. Mesenchymal stem cell therapy for heart disease[J]. Vascul Pharmacol, 2012,57(1): 48-55.

[11] Dai C, Li Y, Yang J, et al. Hepatocyte growth factor preserves beta cell mass and mitigates hyperglycemia in streptozotocin-induced diabetic mice[J]. J Biol Chem, 2003,278(29):27080-27087.

[12] Gao X, Song L, Shen K, et al. Bone marrow mesenchymal stem cells promote the repair of islets from diabetic mice through paracrine actions[J]. Mol Cell Endocrinol, 2014,388(1-2): 41-50.

[13] Castrillo A, Bodelon OG, Bosca L. Inhibitory effect of IGF-I on type 2 nitric oxide synthase expression in Ins-1 cells and protection against activation-dependent apoptosis: involvement of phosphatidylinositol 3-kinase[J]. Diabetes, 2000,49(2):209-217.

[14] Santangelo C, Matarrese P, Masella R, et al. Hepatocyte growth factor protects rat RINm5F cell line against free fatty acid-induced apoptosis by counteracting oxidative stress[J]. J Mol Endocrinol, 2007,38(1-2):147-158.

[15] Hart AW, Baeza N, Apelqvist A,et al. Attenuation of FGF signalling in mouse beta-cells leads to diabetes[J]. Nature, 2000,408(6814):864-868.

Influence of paracrine effects of bone mesenchymal stem cells on proliferation,apoptosis and secretion function of pancreatic β cell line

XUYuxin,JINGQingping,ZHAOCuihong

(TheFirstHospitalofZibo,Zibo255200,China)

Abstract:ObjectiveTo investigate the influence of paracrine effects of bone mesenchymal stem cells (BMSCs) on the proliferation, apoptosis and insulin secretion of pancreatic β cell line INS-1 cells. MethodsThe BMSCs were cultured and BMSCs conditioned media (BMSCs-CM) were obtained by culturing BMSCs with rat pancreatic extracts (RPE) which simulated the pancreatic microenvironment. The INS-1 cells were divided into groups A, B and C, which were cultured with BMSCs-CM, conditioned media without RPE and RPMI 1640 culture solution, respectively. The influence of BMSCs-CM on INS-1 cell proliferation was analyzed by MTT. The cell proliferation rate was calculated. The effects of BMSCs-CM on the INS-1 cell apoptosis were measured by AnnexinⅤ/PI double staining. The rate of apoptosis was counted. The insulin concentration of INS-1 cells was analyzed by radioimmunoassay (RIA).ResultsThe cell proliferation rate in the group A was higher than that in group B and C (P<0.05). The apoptosis rate in the group A was lower than that in group B and C (P<0.05). The insulin concentration in the group A was higher than that in group B and C at the concentration of 5.6 mmol/L and 20.0 mmol/L (all P<0.05).ConclusionsThe incubation of BMSCs-CM can stimulate the INS-1 cell proliferation, inhibit the apoptosis, and promote the insulin secretion. The above actions of BMSCs may be achieved by paracrine effects.

Key words:stem cells, myeloid; pancreatic β cells; cytokines; apoptosis; proliferation

(收稿日期：2015-10-19)

中圖分類號：R587.1

文獻(xiàn)標(biāo)志碼：A

文章編號：1002-266X(2016)14-0001-03

doi：10.3969/j.issn.1002-266X.2016.14.001

通信作者簡介：井慶平(1964-),男，本科，主治醫(yī)師，主要研究方向?yàn)樘悄虿÷圆l(fā)癥的診治。E-mail: jingqingping@sina.com

第一作者簡介：許聿新(1978-),男，博士，主治醫(yī)師，主要研究方向?yàn)樘悄虿÷圆l(fā)癥的診治。E-mail: hongxin1978@sina.com

基金項(xiàng)目：山東省淄博市科學(xué)技術(shù)發(fā)展計(jì)劃項(xiàng)目(2012GG01259)。