CAJ | 학술논문

目的探讨大鼠骨髓间充质干细胞( BMSC)移植对其小肠缺血再灌注损伤的影响.方法获取健康Wistar大鼠骨髓,体外提取、培养BMSC,收获传代培养的第3代细胞并鉴定其表型.在健康Wistar大鼠肠黏膜下注射皮肤黑色素瘤细胞,并用底物荧光素法示踪细胞.另外制作Wistar大鼠肠道缺血再灌注模型,将模型大鼠分为2组:实验组大鼠在肠黏膜下注射BMSC悬液1 ml(含5×106个细胞)；对照组大鼠在肠黏膜下注射等量生理盐水.分别于术后0、2、6、24、72和120h处死大鼠,留取血清和小肠组织样本.采用酶联免疫吸附试验检测血清二胺氧化酶( DAO)、D-乳酸和肿瘤坏死因子α(TNF-α),用光镜和透射电镜观察肠组织,蛋白质印迹法和免疫组织化学法检测紧密连接蛋白-1(ZO-1).结果体外成功分离培养出BMSC.经肠黏膜移植后的皮肤黑色素瘤细胞定植在肠道.实验组大鼠小肠病理改变较对照组轻,小肠黏膜屏障更完好.术后6h实验组DAO为(11.36±1.89) IU/ml,对照组为(14.27±2.09) IU/ml(P＜0.05)；24 h时实验组DAO为(5.04±1.04)IU/ml,对照组为(7.35±1.46) IU/ml(P＜0.05).术后6 h实验组D-乳酸为(1.57±0.25) mmol/L,对照组为(1.93±0.19)mmol/L(P＜0.05)；术后24 h实验组D-乳酸为(1.09±0.13)mmol/L,对照组为(1.41±0.07) mmol/L(P＜0.01).术后6h实验组TNF-α为(266.09±8.84)ng/L,对照组为(286.81±11.54) ng/L (P＜0.01)；术后24 h实验组TNF-α为(190.39±4.24) ng/L,对照组为(218.49±15.51 )ng/L(P＜0.01).实验组ZO-1的表达高于对照组.结论肠黏膜下移植BMSC可以减轻小肠缺血再灌注损伤.
목적 탐토대서골수간충질간세포( BMSC)이식대기소장결혈재관주손상적영향.방법 획취건강Wistar대서골수,체외제취、배양BMSC,수획전대배양적제3대세포병감정기표형.재건강Wistar대서장점막하주사피부흑색소류세포,병용저물형광소법시종세포.령외제작Wistar대서장도결혈재관주모형,장모형대서분위2조:실험조대서재장점막하주사BMSC현액1 ml(함5×106개세포)；대조조대서재장점막하주사등량생리염수.분별우술후0、2、6、24、72화120h처사대서,류취혈청화소장조직양본.채용매련면역흡부시험검측혈청이알양화매( DAO)、D-유산화종류배사인자α(TNF-α),용광경화투사전경관찰장조직,단백질인적법화면역조직화학법검측긴밀련접단백-1(ZO-1).결과 체외성공분리배양출BMSC.경장점막이식후적피부흑색소류세포정식재장도.실험조대서소장병리개변교대조조경,소장점막병장경완호.술후6h실험조DAO위(11.36±1.89) IU/ml,대조조위(14.27±2.09) IU/ml(P＜0.05)；24 h시실험조DAO위(5.04±1.04)IU/ml,대조조위(7.35±1.46) IU/ml(P＜0.05).술후6 h실험조D-유산위(1.57±0.25) mmol/L,대조조위(1.93±0.19)mmol/L(P＜0.05)；술후24 h실험조D-유산위(1.09±0.13)mmol/L,대조조위(1.41±0.07) mmol/L(P＜0.01).술후6h실험조TNF-α위(266.09±8.84)ng/L,대조조위(286.81±11.54) ng/L (P＜0.01)；술후24 h실험조TNF-α위(190.39±4.24) ng/L,대조조위(218.49±15.51 )ng/L(P＜0.01).실험조ZO-1적표체고우대조조.결론 장점막하이식BMSC가이감경소장결혈재관주손상.
Objective To study the effects of bone marrow mesenchymal stem cells (BMSCs) transplantation on the ischemia-reperfusion injury of the intestine in rats.Methods BMSCs were isolated from femur of male Wistar rats and cultured,and the phenotypes of third generation cultured cells were identified.B16-F10-Luc-G5 cells were injected into the intestinal submucosa and traced by Luciferin.Intestinal ischemia-reperfusion injury models were established in male Wistar rats,which were divided into the experimental group (1 ml BMSCs suspension which contained 5 × 106 cells was injected into the intestinal submucosa) and the control group (1 ml normal saline was inject into the intestinal submucosa).Then,serum and intestinal tissue samples were collected at 0,2,6,24,72 and 120 h after operation.Diamine oxidase,D-lactate and TNF-α were tested by ELISA,intestinal tissue samples were observed under the Light microscopy and transmission electron microscopy,and tight junction protein-1 (ZO-1) was detected by using Western blotting and immunohistochemistry.Results BMSCs were isolated and cultured successfully and they colonized in the intestine.The pathological changes of the intestine in experimental group were milder than in control group. Intestinal mucosal barrier was more intact in experimental group than in control group.In the experimental group and control group,DAO was (11.36 ± 1.89) and (14.27 ± 2.09)IU/ml (P＜0.05) at 6th h after injection,and that was (5.04 ± 1.04) and (7.35 ± 1.46) IU/ml (P＜0.05) at 24h after injection,respectively.In the experimental group and control group,D-lactate was (1.57 ± 0.25) and ( 1.93 ± 0.19) mmol/L (P＜0.05) at 6th h after injection,and that was ( 1.09 ± 0.13) and ( 1.41 ± 0.07) mmol/L (P＜0.01 ) at 24th h after injection,respectively.In the experimental group and control group,TNF-α was (266.09 ± 8.84) and (286.81 ± 11.54) ng/L (P＜0.01 ) at 6th h after injection,and that was (190.39 ± 4.24) and (218.49 ± 15.51 )ng/L (P＜0.01 ) at 24th h after injection,respectively.The expression of ZO-1 protein was higher in experimental group than in control group. ConclusionInjection of BMSCs into could protect the intestine from ischemia-reperfusion injury in rats.