中华烧伤杂志
中華燒傷雜誌
중화소상잡지
16
2014年
6期
512-517
,共6页
田甜%贾赤宇%刘毅%刘真%胡国栋%王瑞晨%常春娟
田甜%賈赤宇%劉毅%劉真%鬍國棟%王瑞晨%常春娟
전첨%가적우%류의%류진%호국동%왕서신%상춘연
间质干细胞%移植%脂肪组织%微血管
間質榦細胞%移植%脂肪組織%微血管
간질간세포%이식%지방조직%미혈관
Mesenchymal stem cells%Transplantation%Adipose tissue%Microvessels
目的 观察大鼠同种异体脂肪来源间充质干细胞(ADSC)对自体脂肪颗粒(AG)移植后早期微血管形成的影响. 方法 (1)实验1.取2只SD大鼠双侧腹股沟脂肪组织,采用胶原酶消化法、密度梯度离心法及贴壁法分离、培养、纯化ADSC.取第4代细胞行形态学观察,采用流式细胞仪检测间充质干细胞表面标志物CD34、CD49d、CD106、CD45的表达,并行成脂肪细胞、成骨细胞诱导分化鉴定,噻唑蓝法检测细胞增殖能力.(2)实验2.另取30只SD大鼠,按随机数字表法分为异体AG组6只、自体AG组8只、自体ADSC+自体AG组8只、异体ADSC+自体AG组8只.取4组大鼠一侧腹股沟脂肪组织,自体ADSC+自体AG组制备第4代ADSC,余3组脂肪弃用.取4组大鼠另一侧腹股沟脂肪组织制备自体AG.异体AG组大鼠在移植时将组内大鼠提取的0.6 g AG加入1 mL DMEM/F12培养液中混合后注射移植到另一只大鼠背部皮下,依此类推;自体AG组大鼠注射移植自体AG;自体ADSC+自体AG组大鼠用3.0 ×106个/mL自体ADSC混合液1 mL复合自体AG移植;异体ADSC+自体AG组大鼠采用实验1中2只大鼠提取的3.0×106个/mL ADSC混合液1 mL复合自体AG移植.于移植术后7d取出脂肪移植物,行大体观察、称取湿质量、病理学观察及CD31免疫组织化学染色测定阳性细胞数.对数据行单因素方差分析及SNK检验. 结果 (1)分离培养的第4代细胞形态较均一,与Fb相似;CD34和CD49d呈阳性表达,CD106和CD45呈弱阳性表达;诱导后,细胞可分化为成骨细胞和成脂肪细胞,鉴定为ADSC.第4代ADSC增殖较第10代更快.(2)移植术后7d,4组大鼠脂肪移植物未见明显感染、坏死,异体AG组和自体AG组大鼠脂肪移植物湿质量分别为(0.25±0.04)、(0.26±0.03)g,均小于自体ADSC+自体AG组的(0.36±0.03)g和异体ADSC+自体AG组的(0.35±0.04)g(P值均小于0.05).HE染色结果显示:异体AG组大鼠脂肪移植物脂肪细胞较少,有较多纤维化;自体AG组大鼠脂肪移植物脂肪细胞形态不均一,脂肪细胞周围可见纤维组织;自体ADSC+自体AG组和异体ADSC+自体AG组大鼠脂肪移植物脂肪细胞大小及形态基本一致,未见明显纤维组织.异体AG组大鼠脂肪移植物CD31阳性细胞主要分布在纤维组织,而脂肪细胞周围较少;自体AG组大鼠脂肪移植物CD31阳性细胞分布于脂肪细胞周围,阳性细胞数量远多于异体AG组;自体ADSC+自体AG组和异体ADSC+自体AG组大鼠脂肪移植物CD31阳性细胞数量较自体AG组更多,且主要分布于脂肪细胞周围.自体ADSC+自体AG组和异体ADSC+自体AG组大鼠脂肪移植物每400倍视野下CD31阳性细胞数分别为(20.5±1.1)、(22.1±1.0)个,均较异体AG组和自体AG组的(8.0±3.6)、(10.9±1.7)个多(P值均小于0.05). 结论 异体ADSC复合自体AG移植同自体ADSC复合自体AG移植一样,能够显著促进脂肪移植早期血管化.
目的 觀察大鼠同種異體脂肪來源間充質榦細胞(ADSC)對自體脂肪顆粒(AG)移植後早期微血管形成的影響. 方法 (1)實驗1.取2隻SD大鼠雙側腹股溝脂肪組織,採用膠原酶消化法、密度梯度離心法及貼壁法分離、培養、純化ADSC.取第4代細胞行形態學觀察,採用流式細胞儀檢測間充質榦細胞錶麵標誌物CD34、CD49d、CD106、CD45的錶達,併行成脂肪細胞、成骨細胞誘導分化鑒定,噻唑藍法檢測細胞增殖能力.(2)實驗2.另取30隻SD大鼠,按隨機數字錶法分為異體AG組6隻、自體AG組8隻、自體ADSC+自體AG組8隻、異體ADSC+自體AG組8隻.取4組大鼠一側腹股溝脂肪組織,自體ADSC+自體AG組製備第4代ADSC,餘3組脂肪棄用.取4組大鼠另一側腹股溝脂肪組織製備自體AG.異體AG組大鼠在移植時將組內大鼠提取的0.6 g AG加入1 mL DMEM/F12培養液中混閤後註射移植到另一隻大鼠揹部皮下,依此類推;自體AG組大鼠註射移植自體AG;自體ADSC+自體AG組大鼠用3.0 ×106箇/mL自體ADSC混閤液1 mL複閤自體AG移植;異體ADSC+自體AG組大鼠採用實驗1中2隻大鼠提取的3.0×106箇/mL ADSC混閤液1 mL複閤自體AG移植.于移植術後7d取齣脂肪移植物,行大體觀察、稱取濕質量、病理學觀察及CD31免疫組織化學染色測定暘性細胞數.對數據行單因素方差分析及SNK檢驗. 結果 (1)分離培養的第4代細胞形態較均一,與Fb相似;CD34和CD49d呈暘性錶達,CD106和CD45呈弱暘性錶達;誘導後,細胞可分化為成骨細胞和成脂肪細胞,鑒定為ADSC.第4代ADSC增殖較第10代更快.(2)移植術後7d,4組大鼠脂肪移植物未見明顯感染、壞死,異體AG組和自體AG組大鼠脂肪移植物濕質量分彆為(0.25±0.04)、(0.26±0.03)g,均小于自體ADSC+自體AG組的(0.36±0.03)g和異體ADSC+自體AG組的(0.35±0.04)g(P值均小于0.05).HE染色結果顯示:異體AG組大鼠脂肪移植物脂肪細胞較少,有較多纖維化;自體AG組大鼠脂肪移植物脂肪細胞形態不均一,脂肪細胞週圍可見纖維組織;自體ADSC+自體AG組和異體ADSC+自體AG組大鼠脂肪移植物脂肪細胞大小及形態基本一緻,未見明顯纖維組織.異體AG組大鼠脂肪移植物CD31暘性細胞主要分佈在纖維組織,而脂肪細胞週圍較少;自體AG組大鼠脂肪移植物CD31暘性細胞分佈于脂肪細胞週圍,暘性細胞數量遠多于異體AG組;自體ADSC+自體AG組和異體ADSC+自體AG組大鼠脂肪移植物CD31暘性細胞數量較自體AG組更多,且主要分佈于脂肪細胞週圍.自體ADSC+自體AG組和異體ADSC+自體AG組大鼠脂肪移植物每400倍視野下CD31暘性細胞數分彆為(20.5±1.1)、(22.1±1.0)箇,均較異體AG組和自體AG組的(8.0±3.6)、(10.9±1.7)箇多(P值均小于0.05). 結論 異體ADSC複閤自體AG移植同自體ADSC複閤自體AG移植一樣,能夠顯著促進脂肪移植早期血管化.
목적 관찰대서동충이체지방래원간충질간세포(ADSC)대자체지방과립(AG)이식후조기미혈관형성적영향. 방법 (1)실험1.취2지SD대서쌍측복고구지방조직,채용효원매소화법、밀도제도리심법급첩벽법분리、배양、순화ADSC.취제4대세포행형태학관찰,채용류식세포의검측간충질간세포표면표지물CD34、CD49d、CD106、CD45적표체,병행성지방세포、성골세포유도분화감정,새서람법검측세포증식능력.(2)실험2.령취30지SD대서,안수궤수자표법분위이체AG조6지、자체AG조8지、자체ADSC+자체AG조8지、이체ADSC+자체AG조8지.취4조대서일측복고구지방조직,자체ADSC+자체AG조제비제4대ADSC,여3조지방기용.취4조대서령일측복고구지방조직제비자체AG.이체AG조대서재이식시장조내대서제취적0.6 g AG가입1 mL DMEM/F12배양액중혼합후주사이식도령일지대서배부피하,의차유추;자체AG조대서주사이식자체AG;자체ADSC+자체AG조대서용3.0 ×106개/mL자체ADSC혼합액1 mL복합자체AG이식;이체ADSC+자체AG조대서채용실험1중2지대서제취적3.0×106개/mL ADSC혼합액1 mL복합자체AG이식.우이식술후7d취출지방이식물,행대체관찰、칭취습질량、병이학관찰급CD31면역조직화학염색측정양성세포수.대수거행단인소방차분석급SNK검험. 결과 (1)분리배양적제4대세포형태교균일,여Fb상사;CD34화CD49d정양성표체,CD106화CD45정약양성표체;유도후,세포가분화위성골세포화성지방세포,감정위ADSC.제4대ADSC증식교제10대경쾌.(2)이식술후7d,4조대서지방이식물미견명현감염、배사,이체AG조화자체AG조대서지방이식물습질량분별위(0.25±0.04)、(0.26±0.03)g,균소우자체ADSC+자체AG조적(0.36±0.03)g화이체ADSC+자체AG조적(0.35±0.04)g(P치균소우0.05).HE염색결과현시:이체AG조대서지방이식물지방세포교소,유교다섬유화;자체AG조대서지방이식물지방세포형태불균일,지방세포주위가견섬유조직;자체ADSC+자체AG조화이체ADSC+자체AG조대서지방이식물지방세포대소급형태기본일치,미견명현섬유조직.이체AG조대서지방이식물CD31양성세포주요분포재섬유조직,이지방세포주위교소;자체AG조대서지방이식물CD31양성세포분포우지방세포주위,양성세포수량원다우이체AG조;자체ADSC+자체AG조화이체ADSC+자체AG조대서지방이식물CD31양성세포수량교자체AG조경다,차주요분포우지방세포주위.자체ADSC+자체AG조화이체ADSC+자체AG조대서지방이식물매400배시야하CD31양성세포수분별위(20.5±1.1)、(22.1±1.0)개,균교이체AG조화자체AG조적(8.0±3.6)、(10.9±1.7)개다(P치균소우0.05). 결론 이체ADSC복합자체AG이식동자체ADSC복합자체AG이식일양,능구현저촉진지방이식조기혈관화.
Objective To investigate the effects of allogeneic adipose-derived stem cells (ADSCs) of rat on the early neovascularization of autologous fat transplantation.Methods (1) Experiment 1.Adipose tissue was collected from both inguinal regions of two SD rats to isolate,culture,and purify ADSCs through collagen enzyme digestion,density gradient centrifugation,and adherence method.The fourth passage of cells were collected for morphologic observation,detection of expressions of surface markers CD34,CD49d,CD106,and CD45 of ADSCs with flow cytometer,identification of adipogenic and osteogenic differentiation,and determination of the cell proliferation ability with thiazolyl blue method.(2) Experiment 2.Another 30 SD rats were divided into allogeneic adipose granule (AG) group (A,n =6),autologous AG group (B,n =8),autologous ADSCs + autologous AG group (C,n =8),and allogeneic ADSCs + autologous AG group (D,n =8) according to the random number table.The fourth passage of ADSCs were obtained from adipose tissue from one side of inguinal region of SD rats in group C.Adipose tissue obtained from one side of inguinal region of SD rats of the other 3 groups was abandoned.The AG was prepared from another side of inguinal region of SD rats in the 4 groups.The mixture of 0.6 g AG from one rat and 1 mL DMEM/F12 nutrient solution was injected subcutaneously into the back of another rat in group A,and so on.Autologous AG was injected into its own body of the rats in group B.The mixture of 1 mL autologous ADSCs mixture which contains 3.0 × 106 cells per mililitre autologous ADSCs combined with autologous AG was injected into the rats in group C.The mixture of 1 mL allogeneic ADSCs mixture which contains 3.0 × 106 cells per mililitre ADSCs extractived from the former 2 rats in experiment 1 combined with autologous AG was injected into the rats in group D.At 7 days post transplantation,fat transplants were harvested for gross observation,measurement of wet weight,pathological observation,and assessment of cells with positive expression of CD31 with immunohistochemical method.Data were processed with one-way analysis of variance and SNK test.Results (1) The fourth passage of cells proliferated well showing fusiform shape similar to fibroblasts.These cells showed positive expression of CD34 and CD49d and weak positive expression of CD106 and CD45.They were able to differentiate into adipocytes and osteoblasts.These cells were identified as ADSCs.The fourth passage of cells grew faster than that of the tenth passage.(2) At 7 days post transplantation,no liquifying necrosis or infection was observed in the fat transplants of the rats in the 4 groups.Wet weight of the fat transplants in groups A and B was respectively (0.25 ± 0.04) and (0.26 ± 0.03) g,which were less than those of groups C and D [(0.36 ± 0.03) and (0.35 ± 0.04) g,with P values below 0.05].HE staining showed that there were less fat cells and more fibroblasts in the transplants of group A,visible fibrous tissue around uneven shape of fat cells in the transplants of group B,and almost identical size and shape of fat cells and unobvious fibrous tissues were found in the transplants of groups C and D.The cells with positive expression of CD31 were distributed in fibrous tissues in larger number but less around fat cells in the transplants of group A,while more of these cells were observed surrounding fat cells in the transplants of group B.There were more cells with positive expression of CD31 distributed surrounding fat cells in the transplants of groups C and D than that in group B.The cells with positive expression of CD31 observed under 400 times field were more in number in groups C (20.5 ±1.1) and D (22.1 ±1.0) than in groups A (8.0 ±3.6) andB (10.9±1.7),with P values below 0.05.Conclusions AllogeneicADSCscombined with autologous AG can significantly improve the early vascularization of fat transplantation as well as autologous ADSCs combined with autologous AG.
