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
36期
7108-7112
,共5页
万军%梅举%马金本%马南%单根法
萬軍%梅舉%馬金本%馬南%單根法
만군%매거%마금본%마남%단근법
脐血间充质干细胞%分化%细胞间连接
臍血間充質榦細胞%分化%細胞間連接
제혈간충질간세포%분화%세포간련접
背景:脐血间充质干细胞诱导分化为心肌细胞后移植入心肌缺血组织,可以通过与宿主细胞建立联系来修复取代缺血心肌组织.目的:对犬脐血干细胞进行肌源性诱导,观察其植入后与宿主细胞间的连接情况.设计、时间及地点:随机对照动物实验,于2006-07/2007-10在上海交通大学医学院附属新华医院动物实验中心完成.材料:接近足月的妊娠杂种犬2只,用于分离培养脐血间充质干细胞.成年杂种犬36只,随机数字表法分为细胞移植组、模型对照组,18只/组.方法:取传至第4代的犬脐血间充质干细胞,加入含lacZ基因的重组腺相关病毒载体进行基因转染,继续培养3 d后,行10 μmol/L5-氮杂胞苷肌源性诱导,常规培养3周.两组犬均建立心肌梗死模型,造模后细胞移植组经冠状动脉和局部注射将2 mL细胞悬液(约107个脐血间充质干细胞)移植到梗死区,模型对照组同法注射等量生理盐水.分别于移植后2,4,8周取材制备心肌组织切片,免疫组化检测细胞间连接情况.主要观察指标:脐血间充质干细胞的基因转染、肌源性诱导分化情况,移植细胞与宿主心肌细胞的细胞间连接情况.结果:转染72 h后大部分细胞表达LacZ基因,并合成半乳糖苷酶,X-gal染色呈蓝色.5-aza诱导3周后,脐血干细胞α-Actin(+),Desmin(+),Connexin43(+),而诱导前均呈阴性.移植后第8周,心肌切片中移植细胞和宿主心肌相连处可形成连接,连接处可见cadherin和connexin43绿色荧光阳性表达;模型对照组仅表达cadherin和connexin43,未见带红色荧光的移植细胞.结论:脐血间充质干细胞可在体外诱导为心肌样细胞,移植后能与宿主心肌可能形成有效连接,从而具有通讯联系.
揹景:臍血間充質榦細胞誘導分化為心肌細胞後移植入心肌缺血組織,可以通過與宿主細胞建立聯繫來脩複取代缺血心肌組織.目的:對犬臍血榦細胞進行肌源性誘導,觀察其植入後與宿主細胞間的連接情況.設計、時間及地點:隨機對照動物實驗,于2006-07/2007-10在上海交通大學醫學院附屬新華醫院動物實驗中心完成.材料:接近足月的妊娠雜種犬2隻,用于分離培養臍血間充質榦細胞.成年雜種犬36隻,隨機數字錶法分為細胞移植組、模型對照組,18隻/組.方法:取傳至第4代的犬臍血間充質榦細胞,加入含lacZ基因的重組腺相關病毒載體進行基因轉染,繼續培養3 d後,行10 μmol/L5-氮雜胞苷肌源性誘導,常規培養3週.兩組犬均建立心肌梗死模型,造模後細胞移植組經冠狀動脈和跼部註射將2 mL細胞懸液(約107箇臍血間充質榦細胞)移植到梗死區,模型對照組同法註射等量生理鹽水.分彆于移植後2,4,8週取材製備心肌組織切片,免疫組化檢測細胞間連接情況.主要觀察指標:臍血間充質榦細胞的基因轉染、肌源性誘導分化情況,移植細胞與宿主心肌細胞的細胞間連接情況.結果:轉染72 h後大部分細胞錶達LacZ基因,併閤成半乳糖苷酶,X-gal染色呈藍色.5-aza誘導3週後,臍血榦細胞α-Actin(+),Desmin(+),Connexin43(+),而誘導前均呈陰性.移植後第8週,心肌切片中移植細胞和宿主心肌相連處可形成連接,連接處可見cadherin和connexin43綠色熒光暘性錶達;模型對照組僅錶達cadherin和connexin43,未見帶紅色熒光的移植細胞.結論:臍血間充質榦細胞可在體外誘導為心肌樣細胞,移植後能與宿主心肌可能形成有效連接,從而具有通訊聯繫.
배경:제혈간충질간세포유도분화위심기세포후이식입심기결혈조직,가이통과여숙주세포건립련계래수복취대결혈심기조직.목적:대견제혈간세포진행기원성유도,관찰기식입후여숙주세포간적련접정황.설계、시간급지점:수궤대조동물실험,우2006-07/2007-10재상해교통대학의학원부속신화의원동물실험중심완성.재료:접근족월적임신잡충견2지,용우분리배양제혈간충질간세포.성년잡충견36지,수궤수자표법분위세포이식조、모형대조조,18지/조.방법:취전지제4대적견제혈간충질간세포,가입함lacZ기인적중조선상관병독재체진행기인전염,계속배양3 d후,행10 μmol/L5-담잡포감기원성유도,상규배양3주.량조견균건립심기경사모형,조모후세포이식조경관상동맥화국부주사장2 mL세포현액(약107개제혈간충질간세포)이식도경사구,모형대조조동법주사등량생리염수.분별우이식후2,4,8주취재제비심기조직절편,면역조화검측세포간련접정황.주요관찰지표:제혈간충질간세포적기인전염、기원성유도분화정황,이식세포여숙주심기세포적세포간련접정황.결과:전염72 h후대부분세포표체LacZ기인,병합성반유당감매,X-gal염색정람색.5-aza유도3주후,제혈간세포α-Actin(+),Desmin(+),Connexin43(+),이유도전균정음성.이식후제8주,심기절편중이식세포화숙주심기상련처가형성련접,련접처가견cadherin화connexin43록색형광양성표체;모형대조조부표체cadherin화connexin43,미견대홍색형광적이식세포.결론:제혈간충질간세포가재체외유도위심기양세포,이식후능여숙주심기가능형성유효련접,종이구유통신련계.
BACKGROUND: Umbilical cord blood-mesenchymal stem cells (UCB-MSCs) following differentiation into cardiomyocytes were transplanted into ischemic myocardium. The transplanted cells can build connection with host cells and repair the infarct myocardium. OBJECTIVE: To detect the cell-cell junction after transplantation of the cardiac-like cell derived from the canine umbilical cord blood stem cells. DESIGN, TIME AND SETTING: A randomized controlled animal study was performed from July 2006 to October 2007 at the Animal Experimental Center of Xinhua Hospital Affiliated to School of Medicine, Shanghai Jiao Tong University. MATERIALS: A total of 2 full-term pregnant canines were used for isolation of UCB-MSCs. A total of 36 adult mongrel canines were divided into cell transplantation group and model control group (n=18) according to the rule of random digits table. METHODS: The MSCs at passage 4 were transfected by Laz-Z. After 3-day culture, MSCs were induced by 10 μmol/L 5-azacytidine (5-aza). The canine models of myocardium infarction were established following 3 weeks of culture. 2 mL (1 ×107)MSCs were transplanted into dogs with acute myocardium infarction by coronary artery infusion and local injection in cell transplantation group. An equal volume of saline was used in the model control group. The specimens were harvested and detected at 2, 4 and 8 weeks, respectively. Cell junction was determined using immunohistochemistry. MAIN OUTCOME MEASURES: The following parameters were measured: gene trensfection, myogenic induction and differentiation results of UCB-MSCs; junction of transplanted cells and host cardiomyocytes. RESULTS: Following 72 hours of transfaction, mass of cells expressed LacZ gene, synthetized galactosidase, and stained blue using X-gal staining. Following 3 weeks of 5-aza induction, the antigen a-Actin, Desmin and Connexin43 were all been positively expressed, but before induction they were all negative. From the myocardial section of 8 weeks after transplantation, the junction was formed between the transplanted cells and the host myocardium as formed between the transplanted cells. In the junction, green-fluorescence positive expression of cadherin and connexin43 could be seen. However, in the model control group, only cadherin and connexin43 expressed positively, but the transplanted UCB-MSCs with red fluorescence could not been observed. CONCLUSION: The UCB-MSCs is able to differentiate into cardiac-like cell in vitro and form cell-cell junction in vivo to communicate with surrounding cells.
