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
2009年
23期
4543-4547
,共5页
蒙艳斌%贺莉萍%钱海燕%王歧本%邝满元%潘爱华
矇豔斌%賀莉萍%錢海燕%王歧本%鄺滿元%潘愛華
몽염빈%하리평%전해연%왕기본%광만원%반애화
胚胎干细胞%急性心肌梗死%分化%移植%心肌细胞
胚胎榦細胞%急性心肌梗死%分化%移植%心肌細胞
배태간세포%급성심기경사%분화%이식%심기세포
Embryonic stem cell transplantation in the peripheral infarct site following acute myocardial infarction can prevent ventricular reconstruction%reduce scar area%and improve heart function
背景:现有治疗手段不足以补充梗死心肌,研究表明干细胞移植有可能促使心肌和血管再生,改善心功能和预后.目的:观察急性心肌梗死区中心和周边移植胚胎干细胞后心肌组织形态学及血液动力学的变化.设计、时间及地点:随机对照动物观察,于2007-03/2008-10在中南大学湘雅医学院人体解剖学与神经生物学系神经生物学实验室完成.材料:SPF级Wistar大鼠40只,随机分为4组:正常对照组、梗死模型组、中心移植组、周边移植组,10只/组.胚胎干细胞-D3株(embryonic stem cells-D3,ES-D3)、布法罗大鼠肝细胞均由中国科学院上海细胞所提供.方法:ES-D3细胞复苏后,以(2.0～5.0)× 107L-1种植于培养瓶中,加入含布法罗大鼠肝细胞的条件培养基体外培养分化.除正常对照组外,余3组结扎冠状动脉左前降支建立急性心肌梗死模型.造模后1周接受细胞移植,ES-D3细胞于移植前1 d进行BrdU标记,以胚胎干细胞培养基调整密度至1×109L-1.中心移植组在梗死区选3个点,每点注入10 μL细胞悬液(含104个细胞)至心室壁内;周边移植组在梗死区周边选3个点同法注入等量细胞悬液.主要观察指标:免疫组化及血流动力学指标检测结果.结果:布法罗大鼠肝细胞条件培养基体外培养的ES-D3细胞,呈相对规则的巢状集落样生长,分化8d即可见部分拟胚体出现自发的节律性收缩活动,心肌肌钙蛋白T免疫染色呈阳性,电镜下可见肌管、肌纤维等肌性结构,证实已分化为心肌细胞.周边移植组BrdU免疫荧光染色呈阳性,而中心移植组呈阴性,进一步对BrdU免疫荧光染色阳性细胞行双抗染色,可见心肌肌钙蛋白T呈阳性表达.移植后4周与正常对照组比较,梗死模型组左室收缩、dp/dtmax均减小(P＜0.01),左室舒张末期压上升(P＜0.01),左室质量、左室质量指数均升高(P＜0.01).与梗死模型组比较,中心移植组各项血流动力学指标无明显变化(P＞0.05);周边移植组左室收缩压、±dp/dtmax均显著升高(P＜0.01),左室舒张末期压显著减小(P＜0.01),左室质量、左室质量指数,梗死面积均显著减小(P＜0.01).结论:急性心肌梗死后于梗死周边区移植胚胎干细胞可以阻止心室重构、减少瘢痕面积、改善心功能.
揹景:現有治療手段不足以補充梗死心肌,研究錶明榦細胞移植有可能促使心肌和血管再生,改善心功能和預後.目的:觀察急性心肌梗死區中心和週邊移植胚胎榦細胞後心肌組織形態學及血液動力學的變化.設計、時間及地點:隨機對照動物觀察,于2007-03/2008-10在中南大學湘雅醫學院人體解剖學與神經生物學繫神經生物學實驗室完成.材料:SPF級Wistar大鼠40隻,隨機分為4組:正常對照組、梗死模型組、中心移植組、週邊移植組,10隻/組.胚胎榦細胞-D3株(embryonic stem cells-D3,ES-D3)、佈法囉大鼠肝細胞均由中國科學院上海細胞所提供.方法:ES-D3細胞複囌後,以(2.0～5.0)× 107L-1種植于培養瓶中,加入含佈法囉大鼠肝細胞的條件培養基體外培養分化.除正常對照組外,餘3組結扎冠狀動脈左前降支建立急性心肌梗死模型.造模後1週接受細胞移植,ES-D3細胞于移植前1 d進行BrdU標記,以胚胎榦細胞培養基調整密度至1×109L-1.中心移植組在梗死區選3箇點,每點註入10 μL細胞懸液(含104箇細胞)至心室壁內;週邊移植組在梗死區週邊選3箇點同法註入等量細胞懸液.主要觀察指標:免疫組化及血流動力學指標檢測結果.結果:佈法囉大鼠肝細胞條件培養基體外培養的ES-D3細胞,呈相對規則的巢狀集落樣生長,分化8d即可見部分擬胚體齣現自髮的節律性收縮活動,心肌肌鈣蛋白T免疫染色呈暘性,電鏡下可見肌管、肌纖維等肌性結構,證實已分化為心肌細胞.週邊移植組BrdU免疫熒光染色呈暘性,而中心移植組呈陰性,進一步對BrdU免疫熒光染色暘性細胞行雙抗染色,可見心肌肌鈣蛋白T呈暘性錶達.移植後4週與正常對照組比較,梗死模型組左室收縮、dp/dtmax均減小(P＜0.01),左室舒張末期壓上升(P＜0.01),左室質量、左室質量指數均升高(P＜0.01).與梗死模型組比較,中心移植組各項血流動力學指標無明顯變化(P＞0.05);週邊移植組左室收縮壓、±dp/dtmax均顯著升高(P＜0.01),左室舒張末期壓顯著減小(P＜0.01),左室質量、左室質量指數,梗死麵積均顯著減小(P＜0.01).結論:急性心肌梗死後于梗死週邊區移植胚胎榦細胞可以阻止心室重構、減少瘢痕麵積、改善心功能.
배경:현유치료수단불족이보충경사심기,연구표명간세포이식유가능촉사심기화혈관재생,개선심공능화예후.목적:관찰급성심기경사구중심화주변이식배태간세포후심기조직형태학급혈액동역학적변화.설계、시간급지점:수궤대조동물관찰,우2007-03/2008-10재중남대학상아의학원인체해부학여신경생물학계신경생물학실험실완성.재료:SPF급Wistar대서40지,수궤분위4조:정상대조조、경사모형조、중심이식조、주변이식조,10지/조.배태간세포-D3주(embryonic stem cells-D3,ES-D3)、포법라대서간세포균유중국과학원상해세포소제공.방법:ES-D3세포복소후,이(2.0～5.0)× 107L-1충식우배양병중,가입함포법라대서간세포적조건배양기체외배양분화.제정상대조조외,여3조결찰관상동맥좌전강지건립급성심기경사모형.조모후1주접수세포이식,ES-D3세포우이식전1 d진행BrdU표기,이배태간세포배양기조정밀도지1×109L-1.중심이식조재경사구선3개점,매점주입10 μL세포현액(함104개세포)지심실벽내;주변이식조재경사구주변선3개점동법주입등량세포현액.주요관찰지표:면역조화급혈류동역학지표검측결과.결과:포법라대서간세포조건배양기체외배양적ES-D3세포,정상대규칙적소상집락양생장,분화8d즉가견부분의배체출현자발적절률성수축활동,심기기개단백T면역염색정양성,전경하가견기관、기섬유등기성결구,증실이분화위심기세포.주변이식조BrdU면역형광염색정양성,이중심이식조정음성,진일보대BrdU면역형광염색양성세포행쌍항염색,가견심기기개단백T정양성표체.이식후4주여정상대조조비교,경사모형조좌실수축、dp/dtmax균감소(P＜0.01),좌실서장말기압상승(P＜0.01),좌실질량、좌실질량지수균승고(P＜0.01).여경사모형조비교,중심이식조각항혈류동역학지표무명현변화(P＞0.05);주변이식조좌실수축압、±dp/dtmax균현저승고(P＜0.01),좌실서장말기압현저감소(P＜0.01),좌실질량、좌실질량지수,경사면적균현저감소(P＜0.01).결론:급성심기경사후우경사주변구이식배태간세포가이조지심실중구、감소반흔면적、개선심공능.
BACKGROUND: Present therapeutic tool cannot supplement infarct myocardium. Studies have shown that stem cell transplantation can promote regeneration of myocardium and vessels and improve heart function and prognosis.OBJECTIVE: To observe changes in morphology and hemodynamics in myocardium following embryonic stem cell transplantation in and surrounding the acute myocardial infarct site.DESIGN, TIME AND SETTING: The randomized, controlled, animal study was performed at the Laboratory of Neurobiology,Department of Human Anatomy and Neurobiology, Xiangya Medical College, Central South University from March 2007 to October 2008.MATERIALS: A total of 40 SPF grade Wistar rats were equally randomized into 4 groups, normal control, infarct model,central transplantation and peripheral transplantation groups. Embryonic stem cells-D3 (ES-D3) and Buffalo rat hepatocytas were supplied by Shanghai Cell Institute, Chinese Academy of Sciences.METHODS: Following resuscitation, ES-D3 cells at (2.0-5.0)×107/L were incubated in a flask, and induced to in vitro differentiate in conditioned medium containing Buffalo rat hepatocytes. Except normal control group, rat models of acute myocardial infarction were established by ligating left anterior descending coronary artery in the infarct model, central transplantation and peripheral transplantation groups. At 1 week following model induction, ES-D3 cells were labeled by BrdU for 1 day, and implanted at 1×109/L. Three sites were selected in the infarct site in the central transplantation group. 10 μ L cell suspension (104 cells) was implanted in the ventricular wall through each site. In the peripheral transplantation group, an equal volume of cell suspension was separately implanted in three peripheral infarct sites by the same method.MAIN OUTCOME MEASURES: Results of immunohistochemistry and hemodynamics were measured.RESULTS: ES-D3 cells in buffalo rat hepatocyte conditioned medium presented regular colony-shaped. At 8 days following differentiation, some embryo proper had spontaneous rhythmic contraction, showed positive reaction of cardiac troponin T after immunostaining. Under the electron microscope, myotube and muscle fiber appeared, which verified the differentiation of cardiomyocytes. Cells were positive for BrdU in the peripheral transplantation group, but negative in the central transplantation group. Cells were also positive for cardiac troponin T. 4 weeks following transplantation, left ventricular systolic pressure,minimum/maximum rate of ventricular pressure (±dp/dtmax) were significantly reduced (P ＜ 0.01), but left ventricular end diastolic pressure was significantly increased (P ＜ 0.01), left ventricular mass and left ventricular mass index were significantly increased (P ＜ 0.01 ) in the infarct model group compared with the normal control group. Compared with the infarct model group, no significant changes in hemodynamics indices were found in the central transplantation group (P ＞ 0.05); left ventricular systolic pressure, ±dp/dtmax were significantly increased (P ＜ 0.01), left ventricular end diastolic pressure was significantly decreased (P ＜ 0.01 ), left ventricular mass, left ventricular mass index and infarct area were significantly reduced(P ＜ 0.01) in the peripheral transplantation group.
