中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2008年
29期
5778-5782
,共5页
吴靖芳%薛刚%张耕%王浩宇%郑慧娥%任君旭%吕洋
吳靖芳%薛剛%張耕%王浩宇%鄭慧娥%任君旭%呂洋
오정방%설강%장경%왕호우%정혜아%임군욱%려양
骨髓基质细胞%转分化%免疫细胞化学%胰岛
骨髓基質細胞%轉分化%免疫細胞化學%胰島
골수기질세포%전분화%면역세포화학%이도
背景:已有报道,二甲基亚砜可使骨髓基质细胞分化为神经元,那么可否用二甲基亚砜诱导骨髓基质细胞向nestin阳性细胞分化,继而向胰岛分化.目的:探讨二甲基亚砜对成年大鼠骨髓基质细胞定向诱导分化为胰岛样细胞团的作用.设计:以细胞为观察对象,随机对照,体外实验.单位:河北北方学院医学院组织学与胚胎学教研室.材料:实验于2006-05/2007-07在河北北方学院组胚教研室细胞审完成.选取6～8周龄Wistar大鼠10只,性别不拘,由解放军军事医学科学院实验动物中心提供.实验用分析纯二甲基亚砜购自北京市化学试剂厂,反转录聚合酶链反应系统为Promega公司产品(A3500),Taq酶和DNA marker DL2000购自北京天为时代生物技术有限公司,鼠抗人胰岛素单克隆抗体(ZM20155)、兔抗人胰高血糖素多克隆抗体(zA20119)购自北京中杉金桥生物技术有限公司,兔抗人nestin多克隆抗体(与大鼠组织有良好的交叉反应,可用于检测人、大鼠等哺乳动物组织)、SABC试剂盒购自武汉博士德公司,大鼠胰岛素放射免疫试剂盒,购自美国Linco公司.方法:骨髓基质细胞以含体积分数0.1胎牛血清的H-DMEM培养60 min,收集未贴壁细胞以含10 g/L二甲基亚砜的无血清H-DMEM作为诱导液,以109/cm2密度接种于6孔板,诱导3 d,继之用体积分数0.1胎牛血清的H-DMEM培养7 d.主要观察指标:用DTZ染色观察诱导细胞团的形态,免疫细胞化学染色检测巢蛋白、胰岛素、胰高血糖素、生长抑索的定位:反转录聚合酶链反应检测诱导细胞团的内分泌基因表达;放免分析检测细胞团的胰岛素分泌量.结果:①骨髓基质细胞分化的细胞团形态:骨髓基质细胞为典型的成纤维细胞样或葡萄样贴壁细胞,经二甲基亚砜诱导3 d后,出现胰岛样细胞团,经DTZ染色显示了和胰岛同样的特性.②免疫细胞化学染色显示:二甲基亚砜诱导后1 d骨髓基质细胞表达nestin蛋白,诱导10 d表达胰岛素和胰高血糖素;未经二甲基哑砜诱导的骨髓基质细胞则不表达胰岛素和胰高血糖素.③反转录聚合酶链反应结果显示:骨髓基质细胞经二甲基亚砜诱导后胰岛样细胞团表达insulin1,insulin2,glucagon和somatostain基因.④细胞团的胰岛索分泌量:胰岛样细胞团对葡萄糖刺激敏感,低糖(3.3 mmol/L)和高糖条件下(16.7 mmol/L)胰岛素分泌量分别为5.56和24.5μU/mL.结论:体外经二甲基亚砜诱导的大鼠骨髓基质细胞可定向分化为胰岛样细胞.
揹景:已有報道,二甲基亞砜可使骨髓基質細胞分化為神經元,那麽可否用二甲基亞砜誘導骨髓基質細胞嚮nestin暘性細胞分化,繼而嚮胰島分化.目的:探討二甲基亞砜對成年大鼠骨髓基質細胞定嚮誘導分化為胰島樣細胞糰的作用.設計:以細胞為觀察對象,隨機對照,體外實驗.單位:河北北方學院醫學院組織學與胚胎學教研室.材料:實驗于2006-05/2007-07在河北北方學院組胚教研室細胞審完成.選取6～8週齡Wistar大鼠10隻,性彆不拘,由解放軍軍事醫學科學院實驗動物中心提供.實驗用分析純二甲基亞砜購自北京市化學試劑廠,反轉錄聚閤酶鏈反應繫統為Promega公司產品(A3500),Taq酶和DNA marker DL2000購自北京天為時代生物技術有限公司,鼠抗人胰島素單剋隆抗體(ZM20155)、兔抗人胰高血糖素多剋隆抗體(zA20119)購自北京中杉金橋生物技術有限公司,兔抗人nestin多剋隆抗體(與大鼠組織有良好的交扠反應,可用于檢測人、大鼠等哺乳動物組織)、SABC試劑盒購自武漢博士德公司,大鼠胰島素放射免疫試劑盒,購自美國Linco公司.方法:骨髓基質細胞以含體積分數0.1胎牛血清的H-DMEM培養60 min,收集未貼壁細胞以含10 g/L二甲基亞砜的無血清H-DMEM作為誘導液,以109/cm2密度接種于6孔闆,誘導3 d,繼之用體積分數0.1胎牛血清的H-DMEM培養7 d.主要觀察指標:用DTZ染色觀察誘導細胞糰的形態,免疫細胞化學染色檢測巢蛋白、胰島素、胰高血糖素、生長抑索的定位:反轉錄聚閤酶鏈反應檢測誘導細胞糰的內分泌基因錶達;放免分析檢測細胞糰的胰島素分泌量.結果:①骨髓基質細胞分化的細胞糰形態:骨髓基質細胞為典型的成纖維細胞樣或葡萄樣貼壁細胞,經二甲基亞砜誘導3 d後,齣現胰島樣細胞糰,經DTZ染色顯示瞭和胰島同樣的特性.②免疫細胞化學染色顯示:二甲基亞砜誘導後1 d骨髓基質細胞錶達nestin蛋白,誘導10 d錶達胰島素和胰高血糖素;未經二甲基啞砜誘導的骨髓基質細胞則不錶達胰島素和胰高血糖素.③反轉錄聚閤酶鏈反應結果顯示:骨髓基質細胞經二甲基亞砜誘導後胰島樣細胞糰錶達insulin1,insulin2,glucagon和somatostain基因.④細胞糰的胰島索分泌量:胰島樣細胞糰對葡萄糖刺激敏感,低糖(3.3 mmol/L)和高糖條件下(16.7 mmol/L)胰島素分泌量分彆為5.56和24.5μU/mL.結論:體外經二甲基亞砜誘導的大鼠骨髓基質細胞可定嚮分化為胰島樣細胞.
배경:이유보도,이갑기아풍가사골수기질세포분화위신경원,나요가부용이갑기아풍유도골수기질세포향nestin양성세포분화,계이향이도분화.목적:탐토이갑기아풍대성년대서골수기질세포정향유도분화위이도양세포단적작용.설계:이세포위관찰대상,수궤대조,체외실험.단위:하북북방학원의학원조직학여배태학교연실.재료:실험우2006-05/2007-07재하북북방학원조배교연실세포심완성.선취6～8주령Wistar대서10지,성별불구,유해방군군사의학과학원실험동물중심제공.실험용분석순이갑기아풍구자북경시화학시제엄,반전록취합매련반응계통위Promega공사산품(A3500),Taq매화DNA marker DL2000구자북경천위시대생물기술유한공사,서항인이도소단극륭항체(ZM20155)、토항인이고혈당소다극륭항체(zA20119)구자북경중삼금교생물기술유한공사,토항인nestin다극륭항체(여대서조직유량호적교차반응,가용우검측인、대서등포유동물조직)、SABC시제합구자무한박사덕공사,대서이도소방사면역시제합,구자미국Linco공사.방법:골수기질세포이함체적분수0.1태우혈청적H-DMEM배양60 min,수집미첩벽세포이함10 g/L이갑기아풍적무혈청H-DMEM작위유도액,이109/cm2밀도접충우6공판,유도3 d,계지용체적분수0.1태우혈청적H-DMEM배양7 d.주요관찰지표:용DTZ염색관찰유도세포단적형태,면역세포화학염색검측소단백、이도소、이고혈당소、생장억색적정위:반전록취합매련반응검측유도세포단적내분비기인표체;방면분석검측세포단적이도소분비량.결과:①골수기질세포분화적세포단형태:골수기질세포위전형적성섬유세포양혹포도양첩벽세포,경이갑기아풍유도3 d후,출현이도양세포단,경DTZ염색현시료화이도동양적특성.②면역세포화학염색현시:이갑기아풍유도후1 d골수기질세포표체nestin단백,유도10 d표체이도소화이고혈당소;미경이갑기아풍유도적골수기질세포칙불표체이도소화이고혈당소.③반전록취합매련반응결과현시:골수기질세포경이갑기아풍유도후이도양세포단표체insulin1,insulin2,glucagon화somatostain기인.④세포단적이도색분비량:이도양세포단대포도당자격민감,저당(3.3 mmol/L)화고당조건하(16.7 mmol/L)이도소분비량분별위5.56화24.5μU/mL.결론:체외경이갑기아풍유도적대서골수기질세포가정향분화위이도양세포.
BACKGROUND: Recent findings suggest that bone marrow stromal cells (MSCs) have the capacity to differentiate into neurons induced by dimethyl sulphoxide (DMSO). So we think whether MSCs can trans-differentiate to nestin positive precurosor cells, then to endocrine cells of the pancreas.OBJECTIVE: To investigate the possibility of differentiating functional insulin-producing cells from MSCs induced by DMSO. DESIGN: Randomized and controlled trails for cells in vitro.SETTING: Department of Histology and Embryology, Medical College of Hebei North University. MATERIALS: This study was performed in the Cell Culture Room of Department of Histology and Embryology, Hebei North University from May 2006 to July 2007. Ten SPF Wistar rats of 6-8 weeks age without sex constraint weighing 180-230 g, were purchased from the Center of Experimental Animal Academy of Military Medical Sciences (license: SCXK-armed forces 2002-001). Fetal bovine serum was Gibco products, Trizol and RT-PCR (A3500) kit were purchased from Promega; DMSO from ChemicalReagent Beijing Co., Ltd.; Taq enzyme and DNA marker DL2000 from Tianwei Biotech Beijing Co., Ltd.; mice anti-human insulin monoclonal antibody, rabbit anti-human glucagon and nestin polyclonal antibody from Zhongshan Golden Bridge Biotechnology Co., Ltd.; The polyclonal antibodies had well cross-reaction with the rat's tissue. So they could be used to tissues of human, rats or other mammals. SABC kit was provided by Boster Biotechnology Co., Ltd.; radioimmunoassay kit by Linco Co., Ltd.METHODS: The bone marrow cells woe cultured in H-DMEM supplemented with 0.1 volume fraction of FBS. After 60-minute incubation, non-adherent cells were collected. The cells were re-plated in serum-free DMEM medium at a cell density of 109/cm2 in the presence of 10 mL/L DMSO. The cells were then cultured in DMEM supplemented with 10 mL/L DMSO for 3 days followed by high (25 mmol/L) glucose DMEM media supplemented with 0.1 volume fraction of FBS for 7 additional days. They were plated in plastic six well plates on slide.MAIN OUTCOME MEASURES: The morphology were observed and diphenyl thiocarbazone (DTZ) staining. Immuno-activity was detected using the cytochemical method for the protein expression, such as nestin, insulin, glucagons and somatostatin. The endocrine gene expressions of induced clusters were detected by RT-PCR. Measurement of Insulin content and secretion were detected by immunoassay.RESULTS: The MSC cells woe typically fibrocyte-like, grape-like coherent dnstered cells. They were observed under experiment group alter the 3rd day after DMSO induction, which were similar to pancreatic islet cells. DTZ staining of the cell aggregates were positive as the natural pancreatic islets, Immunocytochernistry also confirmed that these aggregates were positive for nestin on the 1st day after DMSO induction, and positive for insulin, glucagon on the 10th day after DMSO; while the control groups were negatively stained for the characteristics of endocrine. RT-PCR results showed the insulin 1, insulin 2, glucagon and somatostatin geae expressions were on the 7th day after DMSO induction. The aggregates have good reactions to glucose, the secretion of insulin content respectively 5.56 and 24.5 μ U/mL under low glucose (3.3 mmol/L) and high glucose (16.7 mmol/L) conditions.CONCLUSION: The MSCs can be induced to differentiate into pancreatic endocrine hormone-producing cells in serum-flee with DMSO.
