BM-MSC诱导移植免疫耐受和修复慢性移植肾损伤的初步研究
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摘要
目的：探讨骨髓间充质干细胞(BM-MSC)分离、纯化和细胞鉴定的方法和超顺磁氧化铁(SPIO)标记BM-MSC方法的安全性及可行性,分析BM-MSC在体内分布与细胞输注途径的关系,以确定BM-MSC的最佳输注途径。
方法：取西藏小型猪骨髓,采用密度梯度离心法对骨髓间充质干细胞进行分离和纯化,观察其生长曲线。对所获取的细胞进行成脂、成骨诱导分化,并应用流式细胞术检测细胞表面标志物CD45/CD90/CD105,以鉴定所纯化细胞是否为BM-MSC。应用SPIO对BM-MSC进行体外标记,用MTT法检测不同浓度SPIO对标记后细胞活力和增殖能力的影响。建立西藏小型猪同种异体肾移植模型,分别经移植肾动脉和经髂总静脉输注SPIO标记的BM-MSC,切取受体猪各器官,通过普鲁士蓝染色和透射电镜观察标记的BM-MSC在移植肾、肺脏及其他器官的分布情况。
结果：应用梯度密度离心法可获取高纯度的BM-MSC,细胞具有良好的生长活力和传代能力。BM-MSC可经诱导向成骨细胞和脂肪细胞分化,应用茜红素和油红O染色可检测出细胞中钙结节和脂肪颗粒；经流式细胞术检测细胞表面标志物CD90阳性率为96.9%,CD105阳性率为95.7%,CD45阳性率为1.1%(阴性)。SPIO对BM-MSC的标记率接近100%,通过MTT法检测25ug/ml的SPIO对BM-MSC的增殖能力影响较少、又可有效标记细胞是比较适宜的标记浓度,50ug/ml的SPIO标记细胞2天后细胞活性减退。透射电镜可观察到BM-MSC细胞内小囊泡包含的高密度铁粒子。采用供肾动脉与髂总动脉、供肾静脉与髂总静脉端侧吻合的方式成功建立西藏小型猪肾移植模型,将标记的BM-MSC分别经移植肾动脉和髂总静脉输注后,观察BM-MSC在受体猪体内分布显示：经移植肾动脉输注组,BM-MSC在移植肾内定植的细胞数明显多于经髂总静脉输注组,且存在统计学差异；经髂总静脉输注组,肺内的细胞数增加,有统计学差异。两种输注途径均未发生血管栓塞、感染等并发症。
结论：采用密度梯度离心法可获取高纯度、生长活力良好的BM-MSC;利用细胞外形特征、诱导定向分化和细胞表面标志物可成功鉴定BM-MSC;应用SPIO标记BM-MSC的方法简便可行,易于细胞在体内示踪；经移植肾动脉输注BM-MSC是安全和理想的输注途径,有利于BM-MSC在移植肾内定植、分化。
目的：通过对亲属活体肾移植受者输注自体间充质干细胞联合应用低剂量FK506进行免疫诱导,观察移植术后受者的移植肾功能、淋巴细胞亚群及比例和急性排斥反应发生率等指标,探讨自体骨髓间充质干细胞诱导移植免疫耐受的效果及其作用机制。
方法：随机选择2009.9-2012.9在我中心拟行亲属活体供肾移植的慢性肾功能不全(尿毒症期)患者,按照纳入标准入组,分为不输注自体BM-MSC的对照组(10例)和输注白体BM-MSC的实验组(10例)。实验组在术前1月行自体骨髓间充质干细胞制备,分别在术中、术后1周和术后1月共3次输注自体BM-MSC。首次在术中经移植肾动脉输注,细胞数5×106；术后1周和1月经外周静脉输注,细胞数1×106/kg/次。对照组FK506初始剂量为0.08mg.kg-1.d-1,实验组FK506初始剂量为0.04-0.05mg.kg-1.d-1,吗替麦考酚和强的松用量两组相同。术后动态观察移植肾功能、24小时尿蛋白、淋巴细胞计数和比例,应用流式细胞术检测淋巴细胞亚群及比例,并行程序性移植肾穿刺活检,以观察急性排斥反应的发生率等。
结果：采用梯度密度离心法可获取高纯度BM-MSC,其细胞表型为CD44+CD29+CD105+CD48+CD34+。20例受者手术成功,术后未发生移植肾动静脉栓塞、DGF、感染、GVHD等并发症。对照组中1例在移植术后7天发生急性血管性排斥反应并移植肾破裂,实验组未发生急性排斥反应。实验组与对照组均可维持良好的移植肾功能(Cr和Ccr)；实验组在术后早期(1周)24小时尿蛋白定量为308.1+55.1mg,低于对照组727.2±178.1mg, P<0.05,有统计学差异；实验组与对照组淋巴细胞计数和比例、NK细胞的比例、T淋巴细胞亚群CD3+CD4+、CD3+CD8+的计数和CD3+CD4+/CD3+CD8+比值、Treg细胞的比例在术后各时间观察点(术后1周、1月、3月和6月)均无统计学差异,P>0.05。
结论：自体骨髓间充质干细胞联合低剂量FK506能获得理想的移植肾功能,在早期可促进移植肾急性损伤的修复；可获得有效的免疫抑制效果,对淋巴细胞计数和比例、NK细胞的比例、T淋巴细胞亚群计数及比值达到有效调节；可以上调Treg细胞的比例,在一定程度上诱导移植免疫耐受。
目的：通过对慢性移植肾肾病(CAN)的患者输注骨髓间充质干细胞(BM-MSC),观察移植肾功能和移植肾间质纤维化等指标的变化,探讨BM-MSC对慢性移植肾肾病患者移植肾功能的影响和修复慢性移植肾损伤的机制。
方法：选择2009.12-2012.6确诊为慢性移植肾肾病且符合纳入标准的的患者10例,制备第三方健康骨髓间充质干细胞,予CAN患者共三次输注BM-MSC,输注时间为0天、7天和1月。首次在DSA引导下经移植。肾动脉输注,细胞数1.0×106/kg；第2、3次经外周静脉输注,细胞量为5.0×106/kg。临床观察指标包括：Cr、BUN、Ccr、24小时尿蛋白、血/尿β2微球蛋白。在治疗前和治疗后6月行移植肾活检,应用JD图像分析系统检测肾间质纤维化指标(LN、FN、TGF-β、 CTGF)的变化。
结果：除1例患者在第2次输注BM-MSC时出现过敏反应外,未发生出血、移植肾动脉栓塞、假性动脉瘤和感染等与BM-MSC输注相关的并发症。移植肾功能Cr和BUN在治疗前为203.7±46.4umol/L、15.43±8.46mmol/L,治疗后1周、1月分别为185.5±46.2umol/L,12.15±4.96mmol/L和172.6umol/L、11.76±5.12mmol/L,与治疗前比较有下降,差异有统计学意义,P<0.05；而3月及以后的移植肾功能Cr、BUN与治疗前比较,差异无统计学意义。Ccr在治疗后7天、1月、3月分别为43.18±18.04ml/min,43.18±18.04ml/min,48.04±22.34ml/min,较治疗前38.01±12.96ml/min增加,差异有统计学意义,P<0.05。治疗后1月24小时尿蛋白定量为478.5±289.3mg,较治疗前(105.6±327.3mg)减少,差异有统计学意义,P<0.05；治疗后3月及以后则无统计学差异。肾间质纤维化指标FN、LN在治疗6月后的平均光密度值为(0.174±0.027,0.199±0.015)与治疗前(0.182±0.019,0.212±0.013)相比有下降,但差异无统计学意义,P>0.05; TGF-β和CTGF在治疗6月后的平均光密度值为(0.16±0.015,0.139±0.025)与治疗前(0.212±0.017,0.192±0.010)相比有下降,且差异有统计学意义,P<0.05。
结论：慢性移植肾肾病在接受BM-MSC治疗后,血肌酐和尿素氮在1月内均有降低,内生肌酐清除率在3月内有所增加；24小时尿蛋白定量在1月内有所减少；肾间质纤维化相关的指标TGF-β和CTGF的表达减少。
Objective:To investigate the separation, purification and cell identification methods of bone marrow mesenchymal stem cells (BM-MSC), safety and feasibility of labeling BM-MSC by superparamagnetic iron oxide (SPIO). In order to determine the best infusion way of BM-MSC,we analyze the relationship between the distribution of BM-MSC in vivo and cell infusion way.。
Methods:After extraction the bone marrow from Tibetan miniature pig, bone marrow mesenchymal stem cells (BM-MSC) were isolated and purified by the method of density gradient centrifugation, the cell growth characteristics were observed in the light microscope and their growth curves were recorded. Cells obtained were identified by adipogenic, osteogenic induction and surface markers CD45/CD90/CD105, which were detected by flow cytometry. BM-MSC were labeled by SPIO in vitro, the effect of different SPIO concentrations on the viability and proliferation of labeled cells was assayed by MTT method. After Tibetan miniature pig kidney transplantation models were established, SPIO-labeled BM-MSCs were infused separatedly from transplant renal artery and common iliac vein. Organs of the recipient pigs were cut and left for further determination, the distribution of BM-MSC in transplanted kidney, lungs and other organs were observed by biopsies stained by Prussian blue and transmission electron microscopy.
Results:High-purity BM-MSCs with good growth vigor and passage capacity can be obtained by application of gradient density centrifugation method; BM-MSC could be induced to differentiate into bone cells and fat cells, calcium nodules and fat particles can be observed in cells by Qian red pigment and oil red O staining. Cell surface markers were detected by flow cytometry, the positive rate of CD90was96.9%,the positive rate of CD105was95.7%, the positive rate of CD45was1.1%(negative). The rate of BM-MSC labeled by SPIO was close to100%.By MTT assay, we found that25ug/ml was the appropriate and effective label concentration, which had less impact on the proliferative capacity of BM-MSC, significant difference could be found in the proliferative capacity of BM-MSC labeled by SPIO with concentration of50ug/ml after2days. Intracellular vesicles containing high-density iron particles could be observed by transmission electron microscopy. Transplanted models of Tibet mini pig kidneys could be successfully established, end to side anastomosis method was applied to renal artery and common iliac artery, renal vein and common iliac vein. After BM-MSC infused separatedly from transplant renal artery and common iliac vein, we had observed BM-MSC distribution in recepient pigs:BM-MSC cells in renal allografts by the infusing way of transplant renal artery were significantly more than by the way of common iliac vein, there was significant difference, P<0.05; the number of cells in lungs increase by the intravenous infusion way, a statistically significant difference could be found. Complications including blood clots, infection and others had not occurred in all recipient pigs after BM-MSC infused by the two pathway.
Conclusion:High purity, good growth and vitality BM-MSC can be obtained by density gradient centrifugation; BM-MSC can be successfully identified by cell shape characteristics, induction of differentiation and cell surface markers; The method of labeling BM-MSC by SPIO is simple and feasible,it is easier for trace of cells in vivo; Infusion of BM-MSC from transplant renal artery is a safe and ideal pathway,which is conducive for BM-MSC to plant and differentiate in renal allograft.
Objective:To induce immune tolerance by autologous bone marrow mesenchymal stem cells combined with low doses of FK506in living-related donor kidney transplantation; to explore the role and mechanism of induction of transplant immune tolerance by autologous bone marrow mesenchymal stem cells, parameters about immune tolerance will be observed and analyzed such as functions of transplanted kidney, count and rate of lymphocyte subsets, rate of acute rejection and other indicators.
Method:The patients with chronic renal failure (uremia) were enrolled in,who would receive living-related donor kidney transplantation at our center within2009.9to2012.9. The patients,enrolled in according to inclusion criterias, were randomly divided into Group control (10cases, without autologous BM-MSC infusion) and Group experiment (10cases, with autologous BM-MSC infusion). Autologous bone marrow mesenchymal stem cells had been prepared1month before transplantation, BM-MSC would be infused three times in total into patients in Group experiment,the first infusion was during transplantationg, with5×106BM-MSC,by the pathway of transplant renal artery; the second infusion (1week post-transplantation) and the third infusion (1month post-transplantation),with1×106/kg/time BM-MSC cells, by the pathway of peripheral intravenous. The initial dose of FK506for Group control was0.08mg.kg-1.d-1, Group experiment0.04-0.05mg. kg-1.d-1, the same dose of Mycophenolate mofetil and prednisone for the two groups. Functions of renal allograft,24-hour urine protein, lymphocyte count and proportion were dynamically observed after renal transplantation, T lymphocyte subsets and proportion, Treg proportion were detected by flow cytometry, procedural biopsy for transplanted kidneys had been carried out to observe the incidence of acute rejection.
Results:High-purity BM-MSC had been obtained by density gradient centrifugation, cell phenotype was CD44+CD29+CD105+CD48-CD34-, All surgeries of transplantation for20cases were successful without complications such as renal artery and vein thrombosis, DGF, infections and GVHD.1case in Group control had experienced acute vascular rejection and transplant kidney rupture in the7th day after transplantation, there was no1case of AR occurred in Group experiment. Good graft functions (Cr and Ccr) maintained in Group experiment and Group control.24-hour urinary protein excretion of Group experiment in the early period postoperation (1week) was308.1±55.1mg, lower than that (1727.2±178.1mg) of Group control,with significant difference, P<0.05; there were no statistically difference in lymphocyte count and proportion,the count of T lymphocyte subsets(CD3+CD4+T-cell, CD3+CD8+T-cell) and ratio of CD3+CD4+/CD3+CD8+, Treg proportion between Group experimental and Group control at different observation time points post-operation (1week,1month,3months and6months later), P>0.05,
Conclusions:Ideal graft functions can be obtained by autologous bone marrow mesenchymal stem cells combined with low-dose of FK506, it can promote the repair of acute renal injury in the early days. Effective immunosuppressive effect can be induceed by this method, with effective regulation on lymphocyte count and proportion, proportion and ratio of T lymphocyte subsets. It can increase the proportion of Treg cells and induce transplantation tolerance to a certain extent.
Objective:To study the effect of BM-MSC to improve renal allograft functions and the mechanisms of BM-MSC to repair chronic renal allograft injury by comparison of parameters about renal functions and renal interstitial fibrosis of the patients with chronic allograft nephropathy before and after the therapy of bone marrow mesenchymal stem cells.
Methods:10patients with chronic allograft nephropathy (CAN) were diagnosed by inclusion criteria and enrolled in from2009.12to2012.6.Bone marrow mesenchymal stem cells from third-party healthy donor were prepared and infused three times in total, on day0,day7and1month later. The first infusion of BM-MSC was infused by the pathway of transplant renal artery and guided by DSA, the number of cells was1.0×106/kg,2nd and3rd infusion by the pathway of peripheral vein, cell number5.0×106/kg. Clinical indexes including Cr, BUN, Ccr,24-hour urine protein and blood/urine β2-microglobulin were recorded. Procedural allograft biopsy had been unwent before BM-MSC therapy and6months later, JD image analysis system was applied to detect the changes of interstitial fibrosis indicators of renal allograft including LN, FN,TGF-β1and CTGF.
Results:There were no complications such as bleeding, transplant renal artery embolization, pseudoaneurysm and infection,et al,except1case of allergy during the second infusion of BM-MSC.Cr and BUN were203.7±46.4umol/L,15.43±8.46mmol/L before treatment,1week and1month after treatment185.5±46.2umol/L,12.15±4.96mmol/L and172.6umol/L11.76±5.12mmol/L, compared with pre-treatment, the levels of Cr and BUN after therapy had decreased with statistically significant difference (P<0.05). There were no statistical difference between3months and later after therapy and pre-therapy. Ccr of7days,1month and3months after treatmen was43.18±18.04ml/min,43.18in±18.04ml/min,48.04±22.34ml/min respectively, compared with38.01±12.96ml/min pre-treatment, there was statistically significant difference, P<0.05.24hours urinary protein1month after treatment was478.5±289.3mg, less than105.6±327.3mg before treatment,with statistical difference,P<0.05, but without statistical difference in3months and later after treatment and before treatment. The average optical density values of FN and LN,renal interstitial fibrosis markers, were (0.174±0.027,0.199±0.015)6months after treatment,compared with that (0.182±0.019,0.212±0.013) before treatment, the average optical density values had reduced,but without statistical difference,P>0.05; The average optical density values of TGF-β1and CTGF6months after treatment were (0.16±0.015,0.139±0.025), compared with that (0.212±0.017,0.192±0.010) pre-treatment, the average optical density values had reduced with statistical difference, P<0.05.
Conclusions:After BM-MSC treatment, serum creatinine(Cr) and urea nitrogen(Bun) of Chronic allograft nephropathy patients can decrease within1month; endogenous creatinine clearance rate(Ccr) can increase within3months;24-hour urinary protein excretion can reduced within1month; the average optical density values of renal interstitial fibrosis markers TGF-β1and CTGF of renal allograft can reduce.
方法：取西藏小型猪骨髓,采用密度梯度离心法对骨髓间充质干细胞进行分离和纯化,观察其生长曲线。对所获取的细胞进行成脂、成骨诱导分化,并应用流式细胞术检测细胞表面标志物CD45/CD90/CD105,以鉴定所纯化细胞是否为BM-MSC。应用SPIO对BM-MSC进行体外标记,用MTT法检测不同浓度SPIO对标记后细胞活力和增殖能力的影响。建立西藏小型猪同种异体肾移植模型,分别经移植肾动脉和经髂总静脉输注SPIO标记的BM-MSC,切取受体猪各器官,通过普鲁士蓝染色和透射电镜观察标记的BM-MSC在移植肾、肺脏及其他器官的分布情况。
结果：应用梯度密度离心法可获取高纯度的BM-MSC,细胞具有良好的生长活力和传代能力。BM-MSC可经诱导向成骨细胞和脂肪细胞分化,应用茜红素和油红O染色可检测出细胞中钙结节和脂肪颗粒；经流式细胞术检测细胞表面标志物CD90阳性率为96.9%,CD105阳性率为95.7%,CD45阳性率为1.1%(阴性)。SPIO对BM-MSC的标记率接近100%,通过MTT法检测25ug/ml的SPIO对BM-MSC的增殖能力影响较少、又可有效标记细胞是比较适宜的标记浓度,50ug/ml的SPIO标记细胞2天后细胞活性减退。透射电镜可观察到BM-MSC细胞内小囊泡包含的高密度铁粒子。采用供肾动脉与髂总动脉、供肾静脉与髂总静脉端侧吻合的方式成功建立西藏小型猪肾移植模型,将标记的BM-MSC分别经移植肾动脉和髂总静脉输注后,观察BM-MSC在受体猪体内分布显示：经移植肾动脉输注组,BM-MSC在移植肾内定植的细胞数明显多于经髂总静脉输注组,且存在统计学差异；经髂总静脉输注组,肺内的细胞数增加,有统计学差异。两种输注途径均未发生血管栓塞、感染等并发症。
结论：采用密度梯度离心法可获取高纯度、生长活力良好的BM-MSC;利用细胞外形特征、诱导定向分化和细胞表面标志物可成功鉴定BM-MSC;应用SPIO标记BM-MSC的方法简便可行,易于细胞在体内示踪；经移植肾动脉输注BM-MSC是安全和理想的输注途径,有利于BM-MSC在移植肾内定植、分化。
目的：通过对亲属活体肾移植受者输注自体间充质干细胞联合应用低剂量FK506进行免疫诱导,观察移植术后受者的移植肾功能、淋巴细胞亚群及比例和急性排斥反应发生率等指标,探讨自体骨髓间充质干细胞诱导移植免疫耐受的效果及其作用机制。
方法：随机选择2009.9-2012.9在我中心拟行亲属活体供肾移植的慢性肾功能不全(尿毒症期)患者,按照纳入标准入组,分为不输注自体BM-MSC的对照组(10例)和输注白体BM-MSC的实验组(10例)。实验组在术前1月行自体骨髓间充质干细胞制备,分别在术中、术后1周和术后1月共3次输注自体BM-MSC。首次在术中经移植肾动脉输注,细胞数5×106；术后1周和1月经外周静脉输注,细胞数1×106/kg/次。对照组FK506初始剂量为0.08mg.kg-1.d-1,实验组FK506初始剂量为0.04-0.05mg.kg-1.d-1,吗替麦考酚和强的松用量两组相同。术后动态观察移植肾功能、24小时尿蛋白、淋巴细胞计数和比例,应用流式细胞术检测淋巴细胞亚群及比例,并行程序性移植肾穿刺活检,以观察急性排斥反应的发生率等。
结果：采用梯度密度离心法可获取高纯度BM-MSC,其细胞表型为CD44+CD29+CD105+CD48+CD34+。20例受者手术成功,术后未发生移植肾动静脉栓塞、DGF、感染、GVHD等并发症。对照组中1例在移植术后7天发生急性血管性排斥反应并移植肾破裂,实验组未发生急性排斥反应。实验组与对照组均可维持良好的移植肾功能(Cr和Ccr)；实验组在术后早期(1周)24小时尿蛋白定量为308.1+55.1mg,低于对照组727.2±178.1mg, P<0.05,有统计学差异；实验组与对照组淋巴细胞计数和比例、NK细胞的比例、T淋巴细胞亚群CD3+CD4+、CD3+CD8+的计数和CD3+CD4+/CD3+CD8+比值、Treg细胞的比例在术后各时间观察点(术后1周、1月、3月和6月)均无统计学差异,P>0.05。
结论：自体骨髓间充质干细胞联合低剂量FK506能获得理想的移植肾功能,在早期可促进移植肾急性损伤的修复；可获得有效的免疫抑制效果,对淋巴细胞计数和比例、NK细胞的比例、T淋巴细胞亚群计数及比值达到有效调节；可以上调Treg细胞的比例,在一定程度上诱导移植免疫耐受。
目的：通过对慢性移植肾肾病(CAN)的患者输注骨髓间充质干细胞(BM-MSC),观察移植肾功能和移植肾间质纤维化等指标的变化,探讨BM-MSC对慢性移植肾肾病患者移植肾功能的影响和修复慢性移植肾损伤的机制。
方法：选择2009.12-2012.6确诊为慢性移植肾肾病且符合纳入标准的的患者10例,制备第三方健康骨髓间充质干细胞,予CAN患者共三次输注BM-MSC,输注时间为0天、7天和1月。首次在DSA引导下经移植。肾动脉输注,细胞数1.0×106/kg；第2、3次经外周静脉输注,细胞量为5.0×106/kg。临床观察指标包括：Cr、BUN、Ccr、24小时尿蛋白、血/尿β2微球蛋白。在治疗前和治疗后6月行移植肾活检,应用JD图像分析系统检测肾间质纤维化指标(LN、FN、TGF-β、 CTGF)的变化。
结果：除1例患者在第2次输注BM-MSC时出现过敏反应外,未发生出血、移植肾动脉栓塞、假性动脉瘤和感染等与BM-MSC输注相关的并发症。移植肾功能Cr和BUN在治疗前为203.7±46.4umol/L、15.43±8.46mmol/L,治疗后1周、1月分别为185.5±46.2umol/L,12.15±4.96mmol/L和172.6umol/L、11.76±5.12mmol/L,与治疗前比较有下降,差异有统计学意义,P<0.05；而3月及以后的移植肾功能Cr、BUN与治疗前比较,差异无统计学意义。Ccr在治疗后7天、1月、3月分别为43.18±18.04ml/min,43.18±18.04ml/min,48.04±22.34ml/min,较治疗前38.01±12.96ml/min增加,差异有统计学意义,P<0.05。治疗后1月24小时尿蛋白定量为478.5±289.3mg,较治疗前(105.6±327.3mg)减少,差异有统计学意义,P<0.05；治疗后3月及以后则无统计学差异。肾间质纤维化指标FN、LN在治疗6月后的平均光密度值为(0.174±0.027,0.199±0.015)与治疗前(0.182±0.019,0.212±0.013)相比有下降,但差异无统计学意义,P>0.05; TGF-β和CTGF在治疗6月后的平均光密度值为(0.16±0.015,0.139±0.025)与治疗前(0.212±0.017,0.192±0.010)相比有下降,且差异有统计学意义,P<0.05。
结论：慢性移植肾肾病在接受BM-MSC治疗后,血肌酐和尿素氮在1月内均有降低,内生肌酐清除率在3月内有所增加；24小时尿蛋白定量在1月内有所减少；肾间质纤维化相关的指标TGF-β和CTGF的表达减少。
Objective:To investigate the separation, purification and cell identification methods of bone marrow mesenchymal stem cells (BM-MSC), safety and feasibility of labeling BM-MSC by superparamagnetic iron oxide (SPIO). In order to determine the best infusion way of BM-MSC,we analyze the relationship between the distribution of BM-MSC in vivo and cell infusion way.。
Methods:After extraction the bone marrow from Tibetan miniature pig, bone marrow mesenchymal stem cells (BM-MSC) were isolated and purified by the method of density gradient centrifugation, the cell growth characteristics were observed in the light microscope and their growth curves were recorded. Cells obtained were identified by adipogenic, osteogenic induction and surface markers CD45/CD90/CD105, which were detected by flow cytometry. BM-MSC were labeled by SPIO in vitro, the effect of different SPIO concentrations on the viability and proliferation of labeled cells was assayed by MTT method. After Tibetan miniature pig kidney transplantation models were established, SPIO-labeled BM-MSCs were infused separatedly from transplant renal artery and common iliac vein. Organs of the recipient pigs were cut and left for further determination, the distribution of BM-MSC in transplanted kidney, lungs and other organs were observed by biopsies stained by Prussian blue and transmission electron microscopy.
Results:High-purity BM-MSCs with good growth vigor and passage capacity can be obtained by application of gradient density centrifugation method; BM-MSC could be induced to differentiate into bone cells and fat cells, calcium nodules and fat particles can be observed in cells by Qian red pigment and oil red O staining. Cell surface markers were detected by flow cytometry, the positive rate of CD90was96.9%,the positive rate of CD105was95.7%, the positive rate of CD45was1.1%(negative). The rate of BM-MSC labeled by SPIO was close to100%.By MTT assay, we found that25ug/ml was the appropriate and effective label concentration, which had less impact on the proliferative capacity of BM-MSC, significant difference could be found in the proliferative capacity of BM-MSC labeled by SPIO with concentration of50ug/ml after2days. Intracellular vesicles containing high-density iron particles could be observed by transmission electron microscopy. Transplanted models of Tibet mini pig kidneys could be successfully established, end to side anastomosis method was applied to renal artery and common iliac artery, renal vein and common iliac vein. After BM-MSC infused separatedly from transplant renal artery and common iliac vein, we had observed BM-MSC distribution in recepient pigs:BM-MSC cells in renal allografts by the infusing way of transplant renal artery were significantly more than by the way of common iliac vein, there was significant difference, P<0.05; the number of cells in lungs increase by the intravenous infusion way, a statistically significant difference could be found. Complications including blood clots, infection and others had not occurred in all recipient pigs after BM-MSC infused by the two pathway.
Conclusion:High purity, good growth and vitality BM-MSC can be obtained by density gradient centrifugation; BM-MSC can be successfully identified by cell shape characteristics, induction of differentiation and cell surface markers; The method of labeling BM-MSC by SPIO is simple and feasible,it is easier for trace of cells in vivo; Infusion of BM-MSC from transplant renal artery is a safe and ideal pathway,which is conducive for BM-MSC to plant and differentiate in renal allograft.
Objective:To induce immune tolerance by autologous bone marrow mesenchymal stem cells combined with low doses of FK506in living-related donor kidney transplantation; to explore the role and mechanism of induction of transplant immune tolerance by autologous bone marrow mesenchymal stem cells, parameters about immune tolerance will be observed and analyzed such as functions of transplanted kidney, count and rate of lymphocyte subsets, rate of acute rejection and other indicators.
Method:The patients with chronic renal failure (uremia) were enrolled in,who would receive living-related donor kidney transplantation at our center within2009.9to2012.9. The patients,enrolled in according to inclusion criterias, were randomly divided into Group control (10cases, without autologous BM-MSC infusion) and Group experiment (10cases, with autologous BM-MSC infusion). Autologous bone marrow mesenchymal stem cells had been prepared1month before transplantation, BM-MSC would be infused three times in total into patients in Group experiment,the first infusion was during transplantationg, with5×106BM-MSC,by the pathway of transplant renal artery; the second infusion (1week post-transplantation) and the third infusion (1month post-transplantation),with1×106/kg/time BM-MSC cells, by the pathway of peripheral intravenous. The initial dose of FK506for Group control was0.08mg.kg-1.d-1, Group experiment0.04-0.05mg. kg-1.d-1, the same dose of Mycophenolate mofetil and prednisone for the two groups. Functions of renal allograft,24-hour urine protein, lymphocyte count and proportion were dynamically observed after renal transplantation, T lymphocyte subsets and proportion, Treg proportion were detected by flow cytometry, procedural biopsy for transplanted kidneys had been carried out to observe the incidence of acute rejection.
Results:High-purity BM-MSC had been obtained by density gradient centrifugation, cell phenotype was CD44+CD29+CD105+CD48-CD34-, All surgeries of transplantation for20cases were successful without complications such as renal artery and vein thrombosis, DGF, infections and GVHD.1case in Group control had experienced acute vascular rejection and transplant kidney rupture in the7th day after transplantation, there was no1case of AR occurred in Group experiment. Good graft functions (Cr and Ccr) maintained in Group experiment and Group control.24-hour urinary protein excretion of Group experiment in the early period postoperation (1week) was308.1±55.1mg, lower than that (1727.2±178.1mg) of Group control,with significant difference, P<0.05; there were no statistically difference in lymphocyte count and proportion,the count of T lymphocyte subsets(CD3+CD4+T-cell, CD3+CD8+T-cell) and ratio of CD3+CD4+/CD3+CD8+, Treg proportion between Group experimental and Group control at different observation time points post-operation (1week,1month,3months and6months later), P>0.05,
Conclusions:Ideal graft functions can be obtained by autologous bone marrow mesenchymal stem cells combined with low-dose of FK506, it can promote the repair of acute renal injury in the early days. Effective immunosuppressive effect can be induceed by this method, with effective regulation on lymphocyte count and proportion, proportion and ratio of T lymphocyte subsets. It can increase the proportion of Treg cells and induce transplantation tolerance to a certain extent.
Objective:To study the effect of BM-MSC to improve renal allograft functions and the mechanisms of BM-MSC to repair chronic renal allograft injury by comparison of parameters about renal functions and renal interstitial fibrosis of the patients with chronic allograft nephropathy before and after the therapy of bone marrow mesenchymal stem cells.
Methods:10patients with chronic allograft nephropathy (CAN) were diagnosed by inclusion criteria and enrolled in from2009.12to2012.6.Bone marrow mesenchymal stem cells from third-party healthy donor were prepared and infused three times in total, on day0,day7and1month later. The first infusion of BM-MSC was infused by the pathway of transplant renal artery and guided by DSA, the number of cells was1.0×106/kg,2nd and3rd infusion by the pathway of peripheral vein, cell number5.0×106/kg. Clinical indexes including Cr, BUN, Ccr,24-hour urine protein and blood/urine β2-microglobulin were recorded. Procedural allograft biopsy had been unwent before BM-MSC therapy and6months later, JD image analysis system was applied to detect the changes of interstitial fibrosis indicators of renal allograft including LN, FN,TGF-β1and CTGF.
Results:There were no complications such as bleeding, transplant renal artery embolization, pseudoaneurysm and infection,et al,except1case of allergy during the second infusion of BM-MSC.Cr and BUN were203.7±46.4umol/L,15.43±8.46mmol/L before treatment,1week and1month after treatment185.5±46.2umol/L,12.15±4.96mmol/L and172.6umol/L11.76±5.12mmol/L, compared with pre-treatment, the levels of Cr and BUN after therapy had decreased with statistically significant difference (P<0.05). There were no statistical difference between3months and later after therapy and pre-therapy. Ccr of7days,1month and3months after treatmen was43.18±18.04ml/min,43.18in±18.04ml/min,48.04±22.34ml/min respectively, compared with38.01±12.96ml/min pre-treatment, there was statistically significant difference, P<0.05.24hours urinary protein1month after treatment was478.5±289.3mg, less than105.6±327.3mg before treatment,with statistical difference,P<0.05, but without statistical difference in3months and later after treatment and before treatment. The average optical density values of FN and LN,renal interstitial fibrosis markers, were (0.174±0.027,0.199±0.015)6months after treatment,compared with that (0.182±0.019,0.212±0.013) before treatment, the average optical density values had reduced,but without statistical difference,P>0.05; The average optical density values of TGF-β1and CTGF6months after treatment were (0.16±0.015,0.139±0.025), compared with that (0.212±0.017,0.192±0.010) pre-treatment, the average optical density values had reduced with statistical difference, P<0.05.
Conclusions:After BM-MSC treatment, serum creatinine(Cr) and urea nitrogen(Bun) of Chronic allograft nephropathy patients can decrease within1month; endogenous creatinine clearance rate(Ccr) can increase within3months;24-hour urinary protein excretion can reduced within1month; the average optical density values of renal interstitial fibrosis markers TGF-β1and CTGF of renal allograft can reduce.
引文
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