骨髓间充质干细胞及其来源的微泡在慢性肾衰竭中的修复机制

目的:探讨骨髓间充质干细胞及其来源的微泡制备方法及在慢性肾衰竭中的修复机制,为慢性肾衰竭治疗提供方法。方法:①骨髓间充质干细胞制备。选择2只SD大鼠,取大鼠胫骨、股骨髓,利用密度梯度离心法完成单一核细胞分离,采用贴壁方法完成细胞的培养,采用流式细胞仪制备细胞表面特异性抗原,完成细胞的鉴定。②慢性肾衰竭模型制备及处理方法。选择2015年5月～2016年3月医院实验室进行试验的SD大鼠40只,取10只大鼠设为空白对照组,30只大鼠采用肾切除六分之五合并高盐饮食三个月方法建立大鼠慢性肾衰竭模型,随机数字法分为阳性对照组(n=10)、干细胞组(n=10)及微泡干预组(n=10)。阳性对照组建模成功后不采取任何措施处理,干细胞组建模成功后采用移植制备的骨髓间充质干细胞进行治疗,微泡干预组移植骨髓间充质干细胞后采用超声微泡辐照,比较不同组大鼠慢性肾衰竭修复效果。结果:①细胞换液一次细胞纯化呈梭状、纺锤状,以旋涡方式生长,细胞贴壁后生长迅速,细胞传代一次需要3 d,经过三代培养后细胞数量相对较多;②流式细胞仪测定结果表明:对照组细胞表面CD29几乎不表达,而MSC细胞表面CD29阳性率为91. 54%,符合骨髓间充质细胞表面特征;③Western blot法结果表明:HGF蛋白在4组相对分子量为82 000部位存在特异性条带,而EGF蛋白则在4组相对分子量为5 500部位存在特异性条带;④微泡干预组TNF-α水平低于干细胞组与阳性对照组(P<0. 05),微泡干预组细胞黏附分子1水平高于干细胞组与阳性对照组(P<0. 05);⑤阳性对照组建模后未经任何处理,肾脏损伤明显,存在大量炎性细胞;空白对照组未参与建模,肾脏组织完整,未见肾脏组织损伤,干细胞组修复30 d后肾脏损伤缓解,仍存在少许炎性细胞,微泡干预组修复后30 d肾脏组织损伤改善,未见炎性细胞。结论:采用全骨髓法能分离培养骨髓间充质干细胞,将频率为1 MHz、强度1. 0 W/cm2脉冲超声辐照微泡用于慢性肾衰竭大鼠骨髓间充质干细胞修复中能抑制炎性反应。

Preparation of bone marrow mesenchymal stem cells and their bubbles and their repair mechanisms in chronic renal injury

Objective:To investigate the preparation of bone marrow-derived mesenchymal stem cells and their derived microbubbles and the repair mechanism in chronic renal failure,and to provide a method for the treatment of chronic renal failure.Methods:①Preparation of bone marrow mesenchymal stem cells.Two SD rats were selected,and the tibia and femur were harvested from the rats.Mononuclear cells were isolated by density gradient centrifugation.Cells were cultured by adherent method.Flow cytometry was used to prepare cell surface specific antigen for the identification.②Chronic renal failure model preparation and treatment.Forty SD rats were selected from May 2015 to March 2016 in the hospital laboratory.Ten rats were selected as blank control group.Thirty rats were divided into three groups:the high-salt diet model of chronic renal failure in rats was established by monthly method.The rats were randomly divided into positive control group(n=10),stem cell group(n=10)micro-bubble intervention group(n=10).The positive control group did not take any measures after successful modeling.The stem cells were successfully transplanted into the stem cell group for treatment.After the MSCs were transplanted into the micro-bubble intervention group,the ultrasound-induced microbubbles were used for irradiation.Renal failure repair effect.Results:①The primary cell culture of cell renewal was spindle-shaped and spindle-shaped and swirled in vortex.The cell grew rapidly after adhering to the wall.The cell passage required one time for three days and the cell number was relatively more after three generations of culture.②The results showed that there was almost no expression of CD29 on the cell surface of the control group,but the positive rate of CD29 on the surface of MSC was 91.54%and the positive expression of CD29 was positive on the prepared cells.③Western blot showed that the HGF protein was specific in 82 000(P<0.05).④The levels of TNF-αin the micro-bubble intervention group were lower than those in the control group(P<0.05).⑤In the positive control group,the injury of the kidney was obvious and there were a large number of inflammatory cells in the positive control group;the blank control group did not participate in the modeling,and the renal tissue was intact,and no renal tissue injury was observed.After 30 days of repair,the renal damage alleviated and there was still a little inflammatory cells in the stem cell group.After 30 days of repair,dirty tissue repair kidney damage improved,no inflammatory cells.Conclusion:The bone marrow mesenchymal stem cells can be isolated and cultured by the whole bone marrow method and the frequency of 1 MHz,the intensity of 1.0 W/cm 2 pulsed ultrasound irradiation microbubbles for the suppression of inflammatory reaction in bone marrow mesenchymal stem cell repair of chronic renal failure rats.