体外诱导骨髓间充质干细胞向肾小管上皮细胞的分化

Differentiation of bone marrow mesenchymal stem cells into renal tubular epithelial cells in vitro

目的:探讨体外诱导骨髓间充质干细胞向肾小管上皮细胞分化的可行性。方法:实验于2004-10/2005-12在苏州大学儿科研究所和苏州大学附属儿童医院骨科实验室完成。骨髓间充质干细胞特性实验:①将2只4周龄SD大鼠断颈处死,无菌条件下取股骨、胫骨,去除其干骺端,暴露骨髓腔,DMEM培养液冲洗,混匀后用密度为1.077g/L淋巴细胞分离液分离。取单个核细胞层接种50mL细胞培养瓶进行培养,3d后首次换液,待10～14d细胞生长到80%～90%融合时以胰蛋白酶-乙二胺四乙酸消化传代。②采用免疫荧光法测定骨髓间充质干细胞表面CD44与Vimentin的表达情况;应用二苯基四氮唑溴盐法检测细胞生长情况。损伤肾脏组织匀浆诱导骨髓间充质干细胞实验:①取1只成年SD大鼠麻醉后,表皮消毒,取腹部正中切口,寻及双侧肾蒂,用无损伤动脉夹夹闭双侧肾蒂,缺血60min后松开动脉夹,再灌注60min后,无菌取肾制备匀浆。②将肾脏匀浆置于插入式嵌合培养皿中对第3代骨髓间充质干细胞进行诱导,并加入含有5μmol/L全反式维甲酸的最低必须培养基。③诱导第0,3,5,7天,倒置显微镜下观察细胞大体形态变化;以上各时间点取活细胞制成活细胞悬液,经流式细胞仪测定第18型角蛋白的阳性表达率;取诱导分化第7天的细胞,电镜观察其超微结构变化;钙钴法碱性磷酸酶细胞化学染色,与未经损伤肾脏组织匀浆诱导的骨髓间充质干细胞进行比较。结果:①骨髓间充质干细胞的特性检测:免疫荧光法鉴定分离培养的第3代细胞表面CD44和Vimentin表达阳性。二苯基四氮唑溴盐法测得的细胞生长曲线呈倒“S”型。②损伤肾脏组织匀浆诱导后骨髓间充质干细胞情况:骨髓间充质干细胞经诱导后大体形态变圆,由梭形细胞逐渐转变为鹅卵石样细胞,细胞碱性磷酸酶染色呈强阳性。诱导第7天细胞出现微绒毛和紧密连接,经诱导后的骨髓间充质干细胞第18型角蛋白表达阳性率升至79.5%。结论:在缺血再灌注损伤大鼠肾脏匀浆及全反式维甲酸的联合诱导下,骨髓间充质干细胞可向肾小管上皮样细胞分化,具有成为种子细胞应用于急性肾脏损伤治疗的潜在价值。

AIM： To investigate the feasibility of inducing the differentiation of. renal tubular epithelial cells into bone marrow mesenchymal stem cells （BMSCs） in vitro. METHODS： The experiment was conducted in the Laboratory of Orthopedics of Children＇s Hospital Affiliated to Soochow University＇from October 2004 to December 2005. Experiment on BMSC specialty： （1） Two SD rats of 4 weeks old were executed by cutting off the head to sterilely obtain the femoral bone and tibia. The metaphysis was gotten rid off to expose the medullary canal, and DMEMmedium was adopted for washing. The lymphocyte isolation fluid （1.077 g/L） was adopted for isolation after the mixture. The mononuclear cells （50 mL） were inoculated in the cell cutture flask for culture, and the fluid was replaced at 3 days later for the first time. Cells were treated for passage with trypsinisation-edathamil at 10-14 days after 80%-90% of cells were mixed. （2）The CD44 and Vimentin expression on the surface of BMSCs was detected by immumofluorescence staining method, and the cell growth was determined by the 3-（4,5-dimethylthiazol-2-yl）2,5-diphenyl tetrazolium bromide （MTT） assay. Experiment of BMSCs induced by injuried renal tissue homogenate： （1） One adult SD rat was anesthetized and sterilized to make an incision in the middle of abdomen, so as to look for bilateral renal pedicles, which were occluded with non-wound bulldog clamp. The clamp was loosened at 60 minutes after ischemia, and the kidney was obtained sterilely for homogenate following 60-minute reperfusion. （2） The renal homogenate was placed into the snap-on culture dish to induce the BMSCs of the third passage, and 5μmol/L all-trans retinoic acid （ATRA） at a final concentration of 5 μM was added. （3） The morphological changes of the BMSCs were observed under the inverted microscope on the zero, 3^rd, 5= and 7= days of induction, and living cells at each time-point were made into living cell suspension. The flow cytometry was adopted to determine the positive expression rate of cytokeratine 18 （CK 18）. The ultra-structural changes of cells were studied on the 7^th day of differentiation under the electron microscope, and the expression of alkali phosphatase （AKP） enzyme was detected by histochemical staining, which was compared with the BMSCs induced by uninjured renal tissue homogenate. RESULTS： （1） Detection of BMSCs specialty： The BMSCs were positive for CD44 and Vimentin, and the cell growth curve was in an ＇S＇ type. （2） BMSCs induced by injured renal tissue homogenate： Within the days of induction, BMSCs changed from spindle alike cells to cobblestone-like cells. On the 7= day of induction, microvillus and tight junction were observed and AKP staining was positive, which resembled the renal tubular epithelial cell. CK18 expression was greatly enhanced to 79.5% over a period of 7 days. CONCLUSION： BMSCs may undergo epithelial differentiation in vitro under the induction of ischemic reperfusion injured kidney tissue homogenate and ATRA, which might be used as a seed for stem cell therapy for acute renal injury in the future.