CAJ | 학술논문

目的：观察经血红素加氧酶-1（HO-1）基因修饰的骨髓间充质干细胞（BMSCs）在急性肾损伤（AKI）微环境下的增生分化，并探讨其机制。方法Gateway 技术构建含 HO-1目的基因的质粒，同时构建仅含示踪基因 eGFP 的对照载体；脂质体法转染293FT 细胞获得慢病毒原液 lenti-HO-1-eGFP 和 lenti-eGFP；稀释后分别感染 BMSCs 获得 HO-1-BMSCs 和 eGFP-BMSCs（空载体对照），检测转染细胞的活力、分化潜能。制作缺血再灌注诱导 AKI 大鼠的肾脏匀浆上清（AKI-KHS），以正常大鼠肾脏匀浆上清（N-KHS）作为对照，分别干预 BMSCs、eGFP-BMSCs 和 HO-1-BMSCs，构成空白组（BMSCs 组）、对照组（BMSCs／N-KHS 组）、BMSCs／AKI-KHS 组、eGFP-BMSCs／AKI-KHS 组和 HO-1-BMSCs／AKI-KHS 组，于37℃、5％ CO2培养箱中培养3 d。MTT 法检测培养 BMSCs 的生长抑制率，流式细胞仪检测细胞凋亡，透射电镜观察细胞超微结构变化，免疫组化法检测细胞内角蛋白18（CK18）表达，Western 印迹对各组细胞内 HO-1、磷酸化丝氨酸／苏氨酸蛋白激酶（pAKT）、磷酸化细胞外信号调节激酶（pERK）水平进行检测。用 SPSS19．0统计软件进行统计学分析。结果基因修饰并未改变BMSCs 活力及多向分化潜能。与对照组比，BMSCs／AKI-KHS 组的细胞生长抑制率和凋亡阳性细胞比例显著增加（t ＝12．581，t ＝16．283；P ＜0．05）；经 HO-1基因修饰后，HO-1-BMSCs／AKI-KHS 组的细胞生长抑制率和凋亡阳性细胞比例均有显著下降（t ＝5．958，t ＝7．957；P ＜0．05）。AKI-KHS 可诱导BMSCs 出现肾小管上皮细胞样分化的超微结构改变，胞浆中可观察到 CK18表达，以 HO-1-BMSCs／AKI-KHS 组的 CK18＋细胞比例最高（t ＝4．057，P ＜0．05）。与 BMSCs／AKI-KHS 组相比，HO-1-BMSCs／AKI-KHS 组的细胞内 HO-1蛋白水平显著增高（t ＝4．163，P ＜0．05），并伴随着细胞内 pAKT、pERK 蛋白水平显著增高（tpAKT ＝14．305，tpERK ＝7．148；P ＜0．05）。结论 HO-1基因修饰可改善 AKI微环境下培养 BMSCs 的增殖，细胞凋亡减轻，向肾小管上皮样细胞转分化能力增加，HO-1的过表达及其下游的 AKT、ERK 为发挥作用的可能信号通路。目的：觀察經血紅素加氧酶-1（HO-1）基因脩飾的骨髓間充質榦細胞（BMSCs）在急性腎損傷（AKI）微環境下的增生分化，併探討其機製。方法Gateway 技術構建含 HO-1目的基因的質粒，同時構建僅含示蹤基因 eGFP 的對照載體；脂質體法轉染293FT 細胞穫得慢病毒原液 lenti-HO-1-eGFP 和 lenti-eGFP；稀釋後分彆感染 BMSCs 穫得 HO-1-BMSCs 和 eGFP-BMSCs（空載體對照），檢測轉染細胞的活力、分化潛能。製作缺血再灌註誘導 AKI 大鼠的腎髒勻漿上清（AKI-KHS），以正常大鼠腎髒勻漿上清（N-KHS）作為對照，分彆榦預 BMSCs、eGFP-BMSCs 和 HO-1-BMSCs，構成空白組（BMSCs 組）、對照組（BMSCs／N-KHS 組）、BMSCs／AKI-KHS 組、eGFP-BMSCs／AKI-KHS 組和 HO-1-BMSCs／AKI-KHS 組，于37℃、5％ CO2培養箱中培養3 d。MTT 法檢測培養 BMSCs 的生長抑製率，流式細胞儀檢測細胞凋亡，透射電鏡觀察細胞超微結構變化，免疫組化法檢測細胞內角蛋白18（CK18）錶達，Western 印跡對各組細胞內 HO-1、燐痠化絲氨痠／囌氨痠蛋白激酶（pAKT）、燐痠化細胞外信號調節激酶（pERK）水平進行檢測。用 SPSS19．0統計軟件進行統計學分析。結果基因脩飾併未改變BMSCs 活力及多嚮分化潛能。與對照組比，BMSCs／AKI-KHS 組的細胞生長抑製率和凋亡暘性細胞比例顯著增加（t ＝12．581，t ＝16．283；P ＜0．05）；經 HO-1基因脩飾後，HO-1-BMSCs／AKI-KHS 組的細胞生長抑製率和凋亡暘性細胞比例均有顯著下降（t ＝5．958，t ＝7．957；P ＜0．05）。AKI-KHS 可誘導BMSCs 齣現腎小管上皮細胞樣分化的超微結構改變，胞漿中可觀察到 CK18錶達，以 HO-1-BMSCs／AKI-KHS 組的 CK18＋細胞比例最高（t ＝4．057，P ＜0．05）。與 BMSCs／AKI-KHS 組相比，HO-1-BMSCs／AKI-KHS 組的細胞內 HO-1蛋白水平顯著增高（t ＝4．163，P ＜0．05），併伴隨著細胞內 pAKT、pERK 蛋白水平顯著增高（tpAKT ＝14．305，tpERK ＝7．148；P ＜0．05）。結論 HO-1基因脩飾可改善 AKI微環境下培養 BMSCs 的增殖，細胞凋亡減輕，嚮腎小管上皮樣細胞轉分化能力增加，HO-1的過錶達及其下遊的 AKT、ERK 為髮揮作用的可能信號通路。
목적：관찰경혈홍소가양매-1（HO-1）기인수식적골수간충질간세포（BMSCs）재급성신손상（AKI）미배경하적증생분화，병탐토기궤제。방법Gateway 기술구건함 HO-1목적기인적질립，동시구건부함시종기인 eGFP 적대조재체；지질체법전염293FT 세포획득만병독원액 lenti-HO-1-eGFP 화 lenti-eGFP；희석후분별감염 BMSCs 획득 HO-1-BMSCs 화 eGFP-BMSCs（공재체대조），검측전염세포적활력、분화잠능。제작결혈재관주유도 AKI 대서적신장균장상청（AKI-KHS），이정상대서신장균장상청（N-KHS）작위대조，분별간예 BMSCs、eGFP-BMSCs 화 HO-1-BMSCs，구성공백조（BMSCs 조）、대조조（BMSCs／N-KHS 조）、BMSCs／AKI-KHS 조、eGFP-BMSCs／AKI-KHS 조화 HO-1-BMSCs／AKI-KHS 조，우37℃、5％ CO2배양상중배양3 d。MTT 법검측배양 BMSCs 적생장억제솔，류식세포의검측세포조망，투사전경관찰세포초미결구변화，면역조화법검측세포내각단백18（CK18）표체，Western 인적대각조세포내 HO-1、린산화사안산／소안산단백격매（pAKT）、린산화세포외신호조절격매（pERK）수평진행검측。용 SPSS19．0통계연건진행통계학분석。결과기인수식병미개변BMSCs 활력급다향분화잠능。여대조조비，BMSCs／AKI-KHS 조적세포생장억제솔화조망양성세포비례현저증가（t ＝12．581，t ＝16．283；P ＜0．05）；경 HO-1기인수식후，HO-1-BMSCs／AKI-KHS 조적세포생장억제솔화조망양성세포비례균유현저하강（t ＝5．958，t ＝7．957；P ＜0．05）。AKI-KHS 가유도BMSCs 출현신소관상피세포양분화적초미결구개변，포장중가관찰도 CK18표체，이 HO-1-BMSCs／AKI-KHS 조적 CK18＋세포비례최고（t ＝4．057，P ＜0．05）。여 BMSCs／AKI-KHS 조상비，HO-1-BMSCs／AKI-KHS 조적세포내 HO-1단백수평현저증고（t ＝4．163，P ＜0．05），병반수착세포내 pAKT、pERK 단백수평현저증고（tpAKT ＝14．305，tpERK ＝7．148；P ＜0．05）。결론 HO-1기인수식가개선 AKI미배경하배양 BMSCs 적증식，세포조망감경，향신소관상피양세포전분화능력증가，HO-1적과표체급기하유적 AKT、ERK 위발휘작용적가능신호통로。
Objective To investigate the effect of heme oxygenase-1 (HO-1 )gene modification on the proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs)under the acute kidney injury (AKI)microenvironment in vitro and the possible mechanism.Methods Plasmids were constructed by the gateway technology that contained HO-1 target gene (eGFP as the tracing-gene)or only eGFP as the control,and were transfected into the 293FT cells through the liposome method to get the lenti-HO-1 -eGFP/puro and the lenti-eGFP/puro,which then infected BMSCs to obtain HO-1 -BMSCs and eGFP-BMSCs,testing the activity and differentiation potential of transfected cells.The ischemia/reperfusion-AKI kidney homogenate supernatant (KHS)and the control N-KHS were harvested and used to cultivate with BMSCs,eGFP-BMSCs,or HO-1 -BMSCs,respectively,consisting of five groups:the blank group (BMSCs group),control group (BMSCs/N-KHS group),BMSCs/AKI-KHS group,eGFP-BMSCs/AKI-KHS group, and HO-1 -BMSCs/AKI-KHS group,at 37 ℃ in 5% CO2 for 3 days.The MTT method was used to detect the growth inhibitory rate of BMSCs,flow cytometry to detect the cell apoptosis,the transmission electron microscope (TEM)to observe the cell ultrastructure changes,immunohistochemistry to detect the expression of cytokeratin 1 8 (CK1 8),and Western blot to detect the protein expressions of HO-1 ,phospho-AKT (pAKT),and phospho-ERK (pERK).Results The cell viability and differentiation potential of BMSCs were not changed by the gene modification.Compared with the BMSCs/N-KHS group,the growth inhibitory rate of the BMSCs/AKI-KHS group as well as the proportion of apoptotic cells increased significantly (t =1 2.581 ,t =1 6.283,P <0.05),which,after HO-1 gene modification,however,significantly decreased in the HO-1 -BMSCs/AKI-KHS group (t =5.958,t =7.957,P <0.05).AKI-KHS induced ultrastructural change of the renal tubular epithelial differentiation in the cultured BMSCs with CK1 8 expression in the cytoplasm.The proportion of the CK1 8 + cells was the highest in the HO-1 -BMSCs/AKI-KHS group (t =4.057,P <0.05 ).Compared with the BMSCs/AKI-KHS group,the HO-1 -BMSCs/AKI-KHS had significantly increased cellular expression of HO-1 (t =4.1 63,P <0.05),pAKT (tpAKT =1 4.305,P <0.05),and pERK (tpERK =7.1 48;P <0.05).Conclusions In the AKI microenvironment,HO-1 gene modification could improve the proliferation of BMSCs,enhance the ability of BMSCs to differentiate into renal tubular epithelial cells,and decrease the apoptosis of BMSCs.HO-1 overexpression with the downstream AKT and ERK signaling pathway might be the possible mechanism.