CXCR4基因转染人脐带间充质干细胞移植治疗糖尿病肾病的实验研究

摘要 目的：探讨过表达CXCR4的人脐带间充质干细胞（human umbilical cord mesenchymal stem cell, hUC-MSCs）移植后对糖尿病肾病的治疗作用。方法：构建CXCR4的慢病毒表达载体,并建立过表达 CXCR4 的人脐带间充质干细胞（CXCR4-MSCs）。采用8周龄健康雌性SD大鼠75只,其中15只为正常对照组,60只为实验组。实验组糖尿病成模后一个月,将糖尿病实验大鼠60只随机分为4组：①移植CXCR4-MSCs组(CXCR4基因转染MSCs组),即CXCR4组;②移植null-MSCs组(空质粒未转染CXCR4基因的MSCs组),即null-MSCs;③移植MSCs组( MSCs组);④PBS组(未移植任何的MSCs,单纯PBS注射,PBS组)。将CXCR4-MSCs、null-MSCs及MSCs消化离心,取含1×106个细胞悬液经尾静脉分别注入CXCR4-MSCs组、null-MSCs组及MSCs组大鼠体内,PBS组注射l mL PBS。干细胞治疗8周后,处死五组大鼠。各组大鼠处死前放代谢笼留取24 h尿,计算尿量,保存送检。处死前尾静脉采血检测血糖、称体重并记录。观察血糖、肾脏肥大指数、肾重、体重、24小时尿蛋白排泄量,并观察肾脏组织病理学改变。结果：60只SD雌性大鼠糖尿病模型成功率达100％,至实验8周糖尿病大鼠总共死亡14只,存活率达76.67％。实验开始后的8周,所有CXCR4组、Null-MSCs组、MSCs组、PBS组大鼠与正常组比较,体重均明显减轻(P<0.01),血糖明显升高(P<0.01)。MSCs治疗后8周,除正常组外,其余各组大鼠血糖、肾重、肾重/体重比、24小时尿蛋白均显著增高,体重显著降低(P<0.05);与PBS组相比,CXCR4组、null-MSCs组,MSCs组大鼠的肾重、肾重/体重比、24小时尿蛋白均明显降低(P<0.05),体重无明显增加,血糖无明显降低(P>0.05)。CXCR4组大鼠的肾重、肾重/体重比、24小时尿蛋白较除正常组外的各组均明显降低(P<0.05)。糖尿病成模后,给予大鼠尾静脉注射干细胞悬液或等量培养液,注射后8周,除正常组外,其余各组PAS染色可见大鼠肾小球肥大,肾小球基底膜增厚、系膜增生、系膜基质增多,部分肾小球出现明显硬化,符合糖尿病肾病中期病理表现。CXCR4组大鼠肾小球系膜基质增生较其余各组大鼠减少(P<0.05)。结论：转染CXCR4的MSCs可改善糖尿病肾病。

Study on the Role of Lentiviral Expression Vector of CXCR4 Co-transfected with Human Umbilical Cord Mesenchymal Stem Cell in the Treatment of Diabetic Nephropathy

ABSTRACT Objective: To investigate the effects of lentiviral expression vector of CXCR4 co-transfected with human umbilical cord mesenchymal stem cell (hUC-MSCs) transplantation on diabetic nephropathy in rats. Methods: Construct lentiviral expression vector of CXCR4 and construct lentiviral expression vector of CXCR4 co-transfected with hUC-MSCs(CXCR4-MSCs). Animal model: including 15 for normal control group, 60 for the experimental group. Diabetic model was built after one month. 60 diabetic experimental rats were randomly divided into 4 groups: (1) CXCR4 - MSCs group (CXCR4 group); (2) Null - MSCs group (null - MSCs group); (3) MSCs group (MSCs group); (4) PBS group (without any of MSCs transplantation, PBS injection, PBS group). CXCR4 MSCs, null MSCs and MSCs group were received 1×106 MSCs/each by tail vein injection. PBS group was injected l mL PBS. At 8 weeks after treatment, all groups of rats were weighed and put in the 24 h urine metabolism cage to collect urine output before killed. And the tail vein blood glucose was tested. The blood glucose, kidney hypertrophy index weight, body weight, 24 hours urinary protein excretion and renal histopathological changes were observed. Results: Diabetes model success rate was 100%. 60 in SD female rats, 14 diabetic rats were dead at 8weeks, a total survival rate was 76.67%. CXCR4 group, Null - MSCs group, MSCs group, PBS group comparing with the normal group, weight was significantly reduced (P<0.01), glucose was increased significantly (P<0.01). In addition to the normal group, the rest of the group in the rat blood glucose, kidney weight, kidney weight/body weight ratio, 24-hour urinary protein were significantly increased, weight significantly decreased (P<0.05); Compared with the PBS group, kidney weight, kidney weight/body weight ratio, 24-hour urinary protein were significantly lower in CXCR4, null - MSCs group, MSCs group rats (P<0.05), but blood glucose was not decreased (P>0.05). Kidney weight, kidney weight/body weight ratio, 24-hour urine protein of CXCR4 group were significantly lower than Null - MSCs group, MSCs group and the PBS group (P<0.05). In addition to the normal group, the rest of the group was visible in the rat glomerular hypertrophy, GBM thickening, mesangial proliferation and mesangial matrix. Glomerular mesangial matrix proliferation of CXCR4 group was decreased significantly (P<0.05). Conclusion: Transfection of CXCR4 MSCs can be improvement of diabetic nephropathy.