骨髓间充质干细胞防护肾脏衰老的作用及其机制

doi:10.3969/j.issn.2095-4344.2014.19.008

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (19): 2999-3004.doi: 10.3969/j.issn.2095-4344.2014.19.008

• 骨髓干细胞 bone marrow stem cells • 上一篇下一篇

骨髓间充质干细胞防护肾脏衰老的作用及其机制

李艳菊¹，刘洋²，王季石¹，孟伟³，黄懿¹

1贵阳医学院附属医院血液科，贵州省贵阳市 550004
2北京工业大学生命科学与生物医学工程系，北京市 100022
3南方医科大学基因工程研究所，广东省广州市 510515

修回日期:2014-04-17 出版日期:2014-05-07 发布日期:2014-05-07
通讯作者: 李艳菊，贵阳医学院附属医院血液科，贵州省贵阳市 550004
作者简介:李艳菊，博士，副主任医师。
基金资助:
贵州省科技计划基金(黔科合SY[2010]3075)；贵州省卫生厅项目(gzwkj2011-1-023)

Effect and mechanism of mesenchymal stem cells on the aging kidney

Li Yan-ju¹, Liu Yang², Wang Ji-shi¹, Meng Wei³, Huang Yi¹

1Department of Hematology, Affiliated Hospital of Guiyang Medical College, Guiyang 550004, Guizhou Province, China
2Department of Life Science and Biomedical Engineering, Beijing University of Technology, Beijing 100022, China
3Department of Genetic Engineering, Southern Medical University, Guangzhou 510515, Guangdong Province, China

Revised:2014-04-17 Online:2014-05-07 Published:2014-05-07
Contact: Li Yan-ju, Department of Hematology, Affiliated Hospital of Guiyang Medical College, Guiyang 550004, Guizhou Province, China
About author:Li Yan-ju, M.D., Associate chief physician, Department of Hematology, Affiliated Hospital of Guiyang Medical College, Guiyang 550004, Guizhou Province, China
Supported by:
the Science and Technology Plan Fund of Guizhou Province, No. SY[2010]3075; the Grant from the Health Bureau of Guizhou Province, No. gzwkj2011-1-023

摘要/Abstract

摘要：

背景：骨髓间充质干细胞具有分化成肾小管上皮细胞的功能，故推测，间充质干细胞可能具有治疗慢性肾功能减退的作用。
目的：研究骨髓来源的大鼠间充质干细胞防护肾脏衰老的作用及其可能的机制。
方法：实验建立衰老大鼠模型，尾静脉移植骨髓来源的大鼠间充质干细胞，应用荧光染料羟基荧光素二醋酸盐琥珀酰亚胺脂标记体外培养大鼠骨髓来源的间充质干细胞，将其回输肾脏衰老大鼠体内，应用荧光显微镜下观察间充质干细胞是否能够归巢于肾脏；全自动生化仪测定各组大鼠血清中尿素氮及肌酐水平变化；采用免疫荧光法，利用计算机图像叠加技术，观察蓝色荧光4’，6-二脒基-2-苯基吲哚分别与红色荧光的肾小管上皮细胞特异性蛋白-角蛋白的叠加情况。
结果与结论：骨髓来源的大鼠间充质干细胞能够归巢到衰老大鼠的肾脏。与衰老大鼠模型组相比，经过间充质干细胞生物治疗后的衰老大鼠血清中尿素氮及肌酐水平显著降低(P < 0.05)，肾脏组织可见少量4’，6-二脒基-2-苯基吲哚及异硫氰酸荧光素标记的角蛋白双染阳性细胞。结果显示，骨髓来源的大鼠间充质干细胞通过向肾小管上皮分化，这可能是其改善衰老大鼠肾脏的机制之一。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

关键词: 干细胞, 骨髓干细胞, 间充质干细胞, 衰老, 肾脏, 肾小管上皮细胞, 免疫荧光法, D-半乳糖, 角蛋白, 分化

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells can differentiate into renal tubular epithelial cells. It is speculated that mesenchymal stem cells may have a therapeutic role in chronic renal dysfunction.
OBJECTIVE: To study the effect of bone marrow mesenchymal stem cells on the aging kidney of rats and its possible mechanism.
METHODS: Aging models were established in rats. Rat bone marrow mesechymal stem cells cultured in vitro were labeled by 5,6-carboxyfluorescein diacetate, succinimidyl ester, and then injected into the rats via the tail vein. Under a fluorescence microscope, the cell homing was observed. The contents of urea nitrogen and creatinine in serum of rats were tested by automatic biochemical analyzer. The overlaying of blue fluorescent 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride and the specific protein of red fluorescent tubular epithelial cells (keratin protein) was detected by indirect immunofluorescence and computer image stacking technique.
RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells could arrive the kidney of rats. Compared with the model group, the contents of urea nitrogen and creatinine in serum were decreased in the treated group (P < 0.05). The treated group showed a small amount of double positive cells. These findings indicate that rat bone marrow mesenchymal stem cells may differentiate into renal tubular epithelial cells, which may be one of mechanisms that improve the aging kidney of rats.

全文链接：

Key words: renal insufficiency, mesenchymal stem cells, kidney tubules, cell differentiation

中图分类号:

R394.2

李艳菊，刘洋，王季石，孟伟，黄懿 . 骨髓间充质干细胞防护肾脏衰老的作用及其机制[J]. 中国组织工程研究, 2014, 18(19): 2999-3004.

Li Yan-ju, Liu Yang, Wang Ji-shi, Meng Wei, Huang Yi. Effect and mechanism of mesenchymal stem cells on the aging kidney[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(19): 2999-3004.

图/表（结果） 2

2.1 实验动物数量分析 大鼠30只实验中无脱落现象，均进入统计结果分析。

2.2 骨髓间充质干细胞培养及流式细胞仪鉴定的结果 实验采用贴壁法培养的骨髓间充质干细胞光学显微镜下见细胞呈星形、梭形，见图1A，B。

经流式细胞仪检测，结果显示骨髓间充质干细胞的特异性表面标志物：CD45阴性，阳性率为0.83%，CD44阳性，阳性率为92.16%，CD29阳性，阳性率为91.23%。提示培养的细胞主要是骨髓间充质干细胞，见图1D-G。
2.3 骨髓间充质干细胞在肾脏组织的归巢 CFSE为一种绿色荧光染料，可以穿透细胞膜与细胞核中的双链DNA结合而发挥标记的作用，并对细胞的生理活动没有影响，是一种良好的细胞标记物。实验将终浓度为20 μmol/L CFSE染色骨髓间充质干细胞输注模型组大鼠体内24 h后，取出肾脏组织制作冰冻切片，荧光显微镜下观察有荧光细胞存在，见图1C。

2.4 骨髓间充质干细胞移植后大鼠肾脏组织超氧化物歧酶和丙二醛表达 分别于骨髓间充质干细胞细胞移植后第4周处死大鼠，检测正常组肾脏组织中超氧化物歧酶的活性和丙二醛含量分别为：(31.26±2.24) U/mg，(2.756± 0.128) μmol/g；模型组肾脏组织中超氧化物歧酶的活性为(11.47±2.32) U/mg，低于正常组(P < 0.05)，模型组丙二醛含量为(5.367±0.682) μmol/g，高于正常组(P < 0.05)，说明肾脏衰老模型制作成功。

2.5 骨髓间充质干细胞移植后大鼠肾脏组织尿素氮及肌酐的浓度变化 分别于骨髓间充质干细胞细胞移植后第4周处死大鼠，模型组较正常组的尿素氮及肌酐相比呈升高趋势。治疗组尿素氮及肌酐低于模型组(P < 0.05)，见表1。

2.6 骨髓间充质干细胞移植后大鼠肾脏组织角蛋白的表达 分别于骨髓间充质干细胞细胞移植后第4周处死大鼠，通过免疫荧光法，利用计算机图像叠加技术观察到肾脏组织切片，可见蓝色荧光DAPI标记的细胞核内有绿色荧光亮点即FITC标记的角蛋白，说明移植后的骨髓间充质干细胞向肾小管上皮细胞分化。

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引言

材料方法

设计：随机对照动物实验。

时间及地点：于2010年10月至2013年1月在贵阳医学院附属医院生物化学与分子生物学实验室完成。

材料：

动物：选用6-8周龄清洁级SD大鼠30只，雌雄各半，体质量130-150 g，购自贵阳医学院动物实验中心，动物编号：SYXK(黔)2012-0001，动物饲养温度约25 ℃，湿度60%-70%。实验过程中对动物的处置应符合2009年《Ethical issues in animal experimentation》相关动物伦理学标准的条例。

方法：

分组：将30只SD大鼠随机分为分3组，每组10只SD大鼠、雌雄各半。正常组：按SPF动物饲养要求饲养。模型组：每日皮下注射D-半乳糖400 mg/kg，连续注射3个月。按照试剂盒说明书测定正常组和模型组肾脏组织中超氧化物歧酶和丙二醛的水平。治疗组：每日皮下注射D-半乳糖同时，每次以3×10⁶个骨髓间充质干细胞由大鼠尾静脉输入，每周1次，共4次。

骨髓间充质干细胞的培养和鉴定：①将大鼠处死后，无菌条件下取肱骨、股骨和胫骨，并清除附着其上的肌肉组织，将其两端干骺端切除，显露骨髓腔，用2.5 mL注射器吸取PBS反复冲洗骨髓腔，收集细胞悬液1 000 r/min离心3 min后，弃去上清，用含体积分数10%FBS的L-DMDM培养基调整细胞密度，按2×10⁶/cm²密度接种于培养瓶中，置于培养箱中培养。第3天半量换液，第6天用PBS冲洗3遍后全量换液。以后每隔3 d进行1/4-1/5换液，待细胞生长至80%融合时，消化传代，显微镜下观察骨髓间充质干细胞形态。②鉴定：取达到80%-90%融合的生长良好的第4代细胞，胰酶消化后，PBS洗涤一两次，经300目的尼龙网滤过，用PBS调细胞量大约为2×10⁵，制成4个样本，分别加入荧光直标的CD45，CD29，CD44单克隆抗体，室温37 ℃下避光反应30 min，PBS洗涤3次后，应用流式细胞仪检测细胞表面抗原表达，同时用PBS作为一抗设置阴性对照。

骨髓间充质干细胞的肾脏归巢检测：荧光染料CFSE以20 μmol/L的浓度加入骨髓间充质干细胞培养体系中孵育20 min，移植前倒掉培养基，用PBS清洗细胞6次，除去未结合的4’，6-二脒基-2-苯基吲哚(CFSE)，用体积分数1.0%的胰酶消化细胞，PBS清洗2次沉淀后，取经CFSE标记的骨髓间充质干细胞3×10⁵个分别经尾静脉输入衰老大鼠体内，于第24小时处死衰老大鼠，将肾脏组织制作冰冻切片，连续切片15片，片厚30 µm，在荧光显微镜下观察。

大鼠血清中尿素氮及肌酐的浓度变化：治疗结束后第4周，腹腔注射氯胺酮对大鼠进行麻醉，心脏取血，3 000 r/min 离心15 min提取大鼠血清，应用本院临床检验室全自动分析仪采用苦味酸法和脲酶紫外法检测血清中尿素氮、肌酐的含量变化。

免疫荧光法测定骨髓间充质干细胞向肾脏细胞分化：分别于骨髓间充质干细胞移植后第4周处死大鼠，制作肾脏组织冰冻切片，冷丙酮固定冷冻切片30 min，PBS充分冲洗，在中央部位加体积分数0.1% Triton X-100，浸泡20 min，PBS充分冲洗，加体积分数20%的正常血清封闭，置室温10 min，滴加1∶100稀释的多克隆兔抗结蛋白IgG一抗，37 ℃孵育1 h，PBS充分冲洗后，滴加1∶200稀释的异硫氰酸荧光素(FITC)标记的羊抗兔IgG二抗，37 ℃孵育30 min，PBS充分冲洗后，镜检，用PBS代替一抗作阴性对照。实验采用2种荧光标记，植入的细胞用4’，6-二脒基-2-苯基吲哚(DAPI)标记，可在细胞核中表达，角蛋白二抗用异硫氰酸荧光素(FITC)标记，在胞质中表达，DAPI在345 nm紫外光的激发下呈现蓝色荧光，而FITC在488 nm的蓝光激发下呈现绿色荧光；摄取图像，然后利用计算机图像叠加技术，将上述待测靶物质原位显示于同一图像中。

主要观察指标：①间充质干细胞能否归巢于衰老大鼠的肾脏。②间充质干细胞的回输能否降低模型组大鼠尿素氮及肌酐的浓度。③回输的间充质干细胞能否具有向肾小管上皮细胞分化的能力。

统计学分析：所有数据均以x(_)±s表示，应用SPSS 13.0软件进行数据分析，组间均属差异的比较采用两样本t 检验，P < 0.05为差异有显著性意义。

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讨论

衰老是一个自然进程，人到25岁左右时就发育成熟，伴随而来就是人体的衰老^[16-17]。肾脏是功能减退最快的重要器官之一，也是受衰老影响最明显的器官之一。随着人民生活水平的提高，中国老年人群的比重逐年增加，使老年人群慢性肾脏病的发病率明显升高。慢性肾脏病不仅能够导致终末期肾脏疾病，而且能够增加患者心血管疾病的发病率，肾脏衰老正逐渐成为影响国民健康和社会发展的主要疾病。

目前衰老的学说主要有体细胞突变学说、交联学说、自由基学说、神经内分泌学说、免疫学说、细胞凋亡学说和端粒学说等^[18-21]。其中自由基学说占有主流位置，基于自由基学说的原理， D-半乳糖连续皮下注射3个月，根据过多的D-半乳糖在体内氧化产生的大量自由基超过机体的清除能力，引发脂质过氧化的链式反应，产生大量的脂质过氧化物丙二醛，加重对细胞的损伤，引起的代谢紊乱^[22-23]，使机体过氧化反应增强制作肾脏衰老动物模型(即D-半乳糖所致亚急性肾脏衰老模型)，此模型是目前国内外公认的肾脏衰老模型。实验检测模型组与正常组相比，肾脏组织中超氧化物歧酶的活性下降和丙二醛含量升高，说明大鼠肾脏衰老模型制作成功。

间充质干细胞是来源于发育早期中胚层的一类重要的成体干细胞，具有多种组织修复能力，既往大量实验证实：间充质干细胞具有分化为成骨细胞、脂肪细胞、成软骨细胞等细胞的能力。近几年的研究表明，间充质干细胞具有分化成为肾脏细胞的功能，Kale等^[24]选出有表面标记的小鼠间充质干细胞，再以该间充质干细胞移植到经过亚致死剂量放射线照射小鼠体内进行骨髓重建，发现该标记的间充质干细胞可以分化成成熟的肾小管上皮细胞，并参与缺血性肾小管细胞的修复，移植后1周血尿素氮水平正常。Herrera等^[25]给C57/BL6小鼠肌肉注射丙三醇诱导急性肾功能衰竭后灌注标记绿色荧光蛋白的间充质干细胞，发现间充质干细胞集中分布于肾小管上皮并表达细胞角蛋白。故此推测间充质干细胞通过其向肾小管上皮细胞分化而具有治疗肾脏衰老的作用^[26-35]。

间充质干细胞具有以下表型特征：≥95%的细胞表达CD44，CD29，CD105，CD73和CD90等，而绝大多数不表达CD45，CD34，CD14，CD11b，CD79a，CD19等，也不表达MHC II类分子，如HLA-DR抗原等^[36-60]。实验以贴壁法培养大鼠骨髓来源的间充质干细胞，经流式细胞仪检测，CD45阴性、CD44阳性、CD29阳性，故证明所培养的细胞主要是骨髓间充质干细胞。

实验以间充质干细胞作为治疗肾脏衰老的生物工具，通过皮下注射D-半乳糖建立肾脏衰老模型，应用蓝色荧光染料DAPI标记骨髓间充质干细胞，通过尾静脉将标记的骨髓间充质干细胞回输大鼠体内，于骨髓间充质干细胞移植后第4周处死大鼠，制作肾脏组织冰冻切片，采用间接荧光免疫组化法，利用计算机图像叠加技术，实验发现：移植后的骨髓间充质干细胞能够归巢至肾脏，并向肾小管上皮细胞分化，从而达到治疗肾脏衰老的作用。该项研究的深入将多面性地认识骨髓间充质干细胞治疗肾脏衰老的作用机制，祈望骨髓间充质干细胞不仅能够为肾脏提供营养因子，还能够为肾脏补充种子细胞，从而能够从根本上治疗肾脏衰老，避免长期服用药物的弊端，应具有广阔的临床应用前景。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
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骨髓间充质干细胞防护肾脏衰老的作用及其机制

Effect and mechanism of mesenchymal stem cells on the aging kidney

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