骨髓基质干细胞与微粒皮组织促进创面愈合的实验研究
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摘要
[研究背景和目的]
缺乏供皮区和创面愈合时间长是大面积烧伤创面修复的难题,愈合时间延长将使患者机体长期处于消耗状态,从而抵抗力下降、易于发生感染和多器官功能衰竭。自从发明微粒植皮技术后,利用少量的中厚皮片制成微粒皮颗粒,来消灭大面积的创面成为可能。但微粒皮植皮也有其缺点:1.生成的皮肤缺乏完整的真皮结构,较薄非常容易破溃,再次形成创面;2.在微粒皮愈合后创面易产生大量增生瘢痕,影响机体的外观和功能;3.微粒皮本身在创面成活率不确定,有时难以达到理想的效果。
干细胞技术的出现为解决创面修复提供了新的手段,干细胞具有强大的增殖能力,在一定的条件下,可以分化成各种功能细胞。根据发育的阶段,干细胞分为胚胎干细胞和成体干细胞。由于胚胎干细胞的研究受到伦理学方面的困扰,细胞来源受限,因而成体干细胞的研究便受到人们的高度重视。骨髓基质干细胞(Bone mesenchymal stem cell,BMSCs)是一种成体干细胞;BMSCs修复创面的机制目前认为:1.干细胞具有自我更新的能力和强大的分化潜能,能对创伤引起的微环境的改变,根据局部修复的需要分化成所需要的修复细胞并整合到局部组织中;2.在创面局部被激活的BMSCs能够扮演“微型生物反应器”的功能,分泌多种生长因子和细胞因子,调节局部的细胞的增殖分化,细胞基质的形成,血管的形成等过程;3.在机体受到损伤启动炎症反应过程的同时,BMSCs能够在早期迁徙到受损组织参与组织修复。
本课题紧贴临床实际需要,旨在探索应用干细胞技术解决大面积烧伤创面修复这一大难题的途径,依据BMSCs增殖分化的原理,将微粒皮组织与BMSCs一起应用于新鲜创面,运用细胞培养,免疫反应标记、分子生物学、组织病理学微观检测等手段,监测BMSCs增殖分化,以及微粒皮增生爬行的情况。目前国内外尚未开展相关课题的研究。所得数据将为以下方面提供依据:
1.BMSCs在新鲜创而扮演怎样的“微型生物反应器”的功能,其增殖、分化的方向以及形成细胞基质的情况是怎样。
2.在新鲜创面上,BMSCs能否与微粒皮组织黏附发生侵入、迁徙、增殖并分化,促进微粒皮组织的成活。
3.BMSCs与微粒皮组织能否促进创面愈合,提高创面愈合质量。
[方法]
第一部分F344雄性大鼠BMSCs的体外分离培养及鉴定
1.BMSCs分离、培养、传代:断颈处死10～12W F344雄性大鼠,从股骨、胫骨,肱骨中冲洗出骨髓,接种于培养瓶,48h后更换培养液,弃去未贴壁的细胞,每隔48h换液,接近融合的BMSCs按1:2比例传代培养,留取第3代备用。
2.细胞生长动力学:取3、5代细胞MTT法测细胞增殖活性并绘制细胞增殖曲线。
3.BMSCs的细胞周期检测:取传代生长良好的的第3代细胞,0.25%胰酶室温消化后制成细胞悬液,碘化丙啶染色,流式细胞仪检测。
4.流式细胞仪检测细胞表面标记:第3代细胞,0.25%胰酶室温消化后制成细胞悬液,取浓度约1×10~6/ml 1ml分别加入适量FITC标记CD29,CD34,CD44,CD90抗体避光染色,流式细胞仪进行检测。
5.成脂、成骨诱导分化鉴定:第3代细胞,加入成骨、成脂诱导剂进行诱导油红“0”、茜苏红染色鉴定。
第二部分骨髓基质干细胞与微粒皮组织移植创面的实验研究
1.将P5代细胞用5-BrdU 10μmol/L孵育48h标记。
2.取Wistar大鼠全厚皮,剪成4×4cm~2大小作异体皮用。
3.48只F344雌性大鼠随机分为四组:A组骨髓间充质细胞+微粒皮移植组;B组微粒皮移植组;C组BMSCs移植组;D组生理盐水注射组。在F344大鼠背部形成一4×4cm~2创面,留取1/4皮片剪成微粒皮,均匀涂抹在异体皮上,缝合固定创面。
4.于术后14d打开异体皮,观察创面愈合情况,统计创面愈合率,观察并记录创面愈合时间,统计创面收缩率。5.在创面愈合的当日,处死大鼠,留取标本,放入-80℃冰箱保存。
第三部分骨髓基质干细胞在创面增殖、分化情况的实验研究
1.原位杂交
冰冻切片行大鼠Sry基因标记,光学显微镜下检查。
2.免疫荧光组织化学染色
冰冻切片进行BrdU、FⅧ、Keratin、Actin、Nestin、S-100免疫荧光组织化学染色,荧光显微镜下观察。
[结果]
第一部分BMSCs的分离培养扩增及鉴定
1.全骨髓细胞悬液接种于培养瓶后,24h体外培养有少量贴壁细胞,大量的悬浮细胞,48h后弃去悬浮细胞后继续培养,在96h换液后见悬浮细胞减少,细胞呈克隆样生长,10d左右达到细胞融合,基本上没有悬浮细胞。
2.细胞周期:碘化丙啶染色,流式细胞仪检测结果显示:MSCs中的S+G_2+M细胞约占20.19%,而处于G_0/G_1期的细胞为79.81%,大多数为合成期细胞,只有少数的细胞处于活跃的增殖期。
3.BMSCs表面标记的鉴定:CD29细胞阳性率为79.15%,CD34细胞阳性率为9.10%,CD44细胞阳性率为89.47%,CD90细胞阳性率为72.34%。
4.BMSCs成脂、成骨分化潜能的鉴定:成脂诱导14d油红“0”染色,细胞内可见细小脂滴形成,21d脂滴融合成片;成骨诱导14d茜苏红染色,可见少量钙盐沉淀,21d细胞间可见致密的、圆形、成片状的棕红团块,面积较大。
第二部分骨髓基质干细胞与微粒皮组织移植创面的实验研究
1.创面愈合率:伤后14d打开异体皮,A组创面已基本愈合,创面愈合率为85.77±3.47%;B组创面愈合率为62.16±4.39%;C组、D组创面为肉芽创面,创面愈合率分别为30.41±1.73%,29.96±1.33%,创面的愈合是因为创面的收缩。Welch=1028.700,P＜0.001,A、B两组比较有显著性差异P＜0.001,C、D两组比较没有显著性差异,P=0.898。
2.创面愈合时间:A组的创面愈合时间为17.17±1.64;B组为20.33±2.38;C组为23.08±1.31;D组为25.16±1.53。F=46.372,P＜0.001,A、B两组比较有显著性差异P＜0.001,C、D两组比较有显著性差异,P=0.006。
3.创面收缩率:A组的创面收缩率为36.89±1.94%;B组为39.56±2.93%;C组为92.15±1.44%;D组为92.40±1.91%;F=2608.977,P＜0.001,A、B两组比较有显著性差异P＜0.01,C、D两组比较没有显著性差异,P=0.903。
第三部分骨髓基质干细胞在创面增殖、分化情况的实验研究
1.原位杂交:A组微粒皮标本切片上,在毛囊的周围可见较多的,细胞核为棕色的细胞,真皮层内可见散在的核为棕色的细胞。C组标本在愈合瘢痕组织内可见散在的核为棕色的细胞。B组、D组标本未见核为棕色的细胞。
2.免疫组化双荧光染色:
1) A组标本可见较强的标记BrdU的红色荧光和Keratin的绿色荧光出现在毛囊周围,并且位于同一位置;B组标本可见较强的绿色荧光位于上皮基底层,但未见到相应BrdU标记的红色荧光。D组标本未见两种颜色的荧光。
2) A组与C组在真皮层内可见血管样结构的标记BrdU的红色荧光和FⅧ的绿色荧光位于同一位置;B组标本在真皮层内可见散在的绿色荧光,D组标本在愈合瘢痕组织可见散在的绿色荧光,但两组均未见相应的标记BrdU的红色荧光出现。
3) A、C两组在真皮层内可见散在的标记BrdU的红色荧光和Actin的绿色荧光位于同一位置;B组标本在真皮层内可见散在的绿色荧光,D组标本在愈合瘢痕组织可见散在的绿色荧光,但两组均未见相应的标记BrdU的红色荧光出现。
4) A、C两组在真皮层内可见散在的标记BrdU的红色荧光和Nestin的绿色荧光位于同一位置。B、D组标本未见两种颜色的荧光。
5) A、C两组在真皮层内可见散在的标记BrdU的红色荧光和Nestin的绿色荧光位于同一位置。B、D组标本未见两种颜色的荧光。
[结论]
1.成功地从大鼠骨髓中分离出BMSCs,并证实分离的BMSCs具有多项分化潜能,符合成体干细胞的特性
2.移植创面的BMSCs能促进微粒皮组织成活,并促进创面愈合
3.移植于创面的BMSCs在创面及微粒皮组织微环境的作用下,根据创面修复的需要,可能分化成为血管内皮细胞、肌成纤维细胞、表皮细胞,周围神经组织细胞。
[Background and Objective]
The two difficult problems of severe burns are Lack of skin donor sites and delayed time of skin wound healing,delayed time of skin wound healing can result in a consuming state of body in a long period.This can lead to the descent of resistance abilities of body,easy getting infections and multiple organ failure.Since the invention of microskin transplantation technique,utilizing a small quantity of splitthickness skin graft to make into miroskin tissue particles,the extinction of wound surface be become into reality.This bring a revolutionary breakthrough in repairing severe burns wound surface. There are some shortcomings of microskin transplantation in this technique:1.The skin by microskin extended is lack of integral dermis, very thin and easy to ulcerate,be prone to become wound surface again; 2.After the wound surface healing of applying microskin tissue particles transplantation,a mass of hyperplasia scar come into being, affected the appearance and function of the body.3.The surviving of microskin on wound surface is not insure some time,and will not get a good result.
The coming forth of stem cell technique bring a new way in repairing the wound surface.The stem cell can be provided with strong hyperplastic ability.In a certain condition,it can be differentiated into kinds of functional cells.Base on the phase of cell development,stem cell be divided into two categories:embryonic stem cell and adult stem cell. The research in embryonic stem cells is restricted by its limited source and ethical controversy,so research on adult stem cells attracts more attentions.Bone marrow mesenchymal stem cell is a kind of adult stem cell,derived from mesoderm.The mechanism of BMSCs repairing wound surface lie in the following ways:1.Stem cells have self-regenerate ability and strong differentiate ability.Adapt to the microenvironment change on wound,it can be differentiated into kinds of cells and integrated into local tissues according to the needs of the body.2.The enabled BMSCs in wound surface can act as a micro-bioreactor,it can secrete kinds of growth factor and cell factor,accommodate the proliferation and differentiation of local tissue,the formation of cell matrix,the formation of vascular et al;3.Once the body has been damaged, at the same time of starting process of inflammation reaction,BMSCs can migrate into damaged tissue and join the repairing of the tissue.
This project bases on the principle of the proliferation and differentiation of BMSCs.Transplanted the BMSCs and microskin tissue into fresh wound surface,utilize cell culture immunological marking and reaction,molecular biology histopathology microcosmic detection, monitoring the proliferation and differentiation of BMSCs and the hyperplasia and extending of microskin particles,The experimental data will provide the evidences for the following way:
1.How are the BMSCs acted as micro-bioreactor in fresh wound surface, and how are the orientation of proliferation and differentiation and the way of forming cell matrix.
2.Whether BMSCs can attach to and invade into and migrate into microskin tissue particles,proliferating and differentiating,and improve the surviving of microskin tissue particles in fresh wound surface.
3.Whether the BMSCs and microskin tissue particles can accelerate the healing of wound surface,and improve the healing quality of wound surface.
[Methods]
Part one Isolation and identification of BMSCs of male F344 rat in vitro
1.Isolation,culture and passage:break off the neck and execute male F344 rat,douche the bone marrow from femur,tibia and humerus, inoculate these cells into culture flask,replace the culture media after 48 hours,remove the non-adherent cells.Culture media was changed every 48 hours.Cells were passaged with ratio of 1:2 when almost confluent.Passage 3 cells were used for further experiment.
2.Cell growth kinetics:cell proliferation activity of passage 3 and passage 5 cells were measured,cell proliferation curve were be drawn.
3.Cell cycle examination of BMSCs:active growing passage 3 cells were detached by 0.25%trypsin and made into single cell suspension,stained for propidium iodide,then passed through flow cytometer.
4.Cell surface marker examination of BMSCs by flow cytometer:active growing passage 3 cells were detached by 0.25%trypsin and made into single cell suspension,take 1ml cells which concentration is 1×10~6/ml and stain for CD29,CD34,CD44 and CD90 labelled with FITC,then measured by flow cytometer.
5.Lipogenesis and osteogenesis induction and characterization: passaged 3 cells were induced with lipogenetic reagent and osteogenetic reagent,stained for Oil Red 0 and Alizarin Red S, measured by microscope.
Part two experimental study on transplantation of bone mesenchymal stem cell and microskin tissue into wound surface
1.take passage 5 cells labeled with 5-Brdu(10μmol/L) for 48 hours.
2.Take full thickness skin graft of Wistar rat,cut into size 4×4cm~2
3.48 F344 female rat take into four groups:group A transplant bone mesenchymal stem cell and microskin tissue particles into wound surface;group B transplant microskin tissue particles into wound surface;group C transplant bone mesenchymal stem cell into wound surface;group D injected with physiological saline solution.Forming a wound surface of size 4×4cm~2,take a quarter of full thickness skin graft cut into microskin tissue particles,lay on surface of xenoma skin,suture and fix on the surface.
4.Opened up the xenoma skin 14 days post-operation,observed and record healing rate of wound surface;Observed and recorded healing time of wound surface;Record the shrinkage rate of wound surface.
5.On the day of wound surface healing,execute the rat,took the skin specimen,preserved the specimen on refrigerator of -80℃.
Part three experimental study on proliferation and differentiation of the bone mesenchymal stem cell on wound surface
1.hybridization in situ
Rat sry gene was labelled in freezing section,measured in optical microscope.
2.Examination of immune fluorescence histochemistry
Freezing sections were stained for Cy3 conjugated secondary antibody combined with BrdU antibody,FITC conjugated secondary antibody combined with FⅧ,Keratin,actin,nestin and S-100 antibody,analysed with fluorescence microscope.
[Result]
Part one Isolation and identification of BMSCs of male F344 rat in vitro
1.After the full bone marrow cell suspension was inoculated in a culture flask,a small amount of bone marrow mononuclear cells adhere to the flask 24 hours after planting.After 48 hours,suspending cells were discarded.In 96 hours,suspending cells were decreased obviously,the cells were be growing with clone formation,reaching homogeneous confluence in 12 hours.In 10 days,the cells were fusion,cannot see the suspending cells basically.
2.Flow cytometer analysis showed that about 20.19%of cells are in S+G2+M phase,79.81%are in Go/G phase,and only small numbers are in active proliferation state.
3.Identification of BMSCs superficial marker:79.15%cells were CD29 positive cells;9.10%cells were CD34 positive cells;89.47%cells were CD44 positive cells;72.34%cells were CD90 positive cells.
4.lipogenesis and osteogenesis induction and characterization:After 14 days lipogenesis induction,and stained for Oil Red O,many tiny lipid droplet can be seen in cytoplasm,In 21 days,lipid droplet in cytoplasm fused,like vacuole,osteogenesis induction stained for Alizarin Red S,in 14 days,calcium salt deposition can be seen;In 21 days,brown red mass can be seen among the induction cells.
Part two experimental study on transplantation of bone mesenchymal stem cell and microskin tissue particles into wound surface
1.Healing rate of wound surface:xenoma skin was opened in 14 days,wound surface of group A had been healed basically,healing rate of wound surface was 85.77±3.47%;healing rate of wound surface was 62.16±4.39%;The wound surface of group C,group D was flesh bud tissue healing rate of wound surface was 30.44±1.73%,29.96±1.33% respectively;The healing of wound surface was own to shrinkage of wound surface.Comparison between group A and group B existed in Significant difference(P＜0.001);Welch=1028.700,P＜0.001;Comparison between group C and group D has no difference(P=0.898)
2.Healing time of wound surface:The healing time of wound surface of four groups is 17.17±1.64,20.33±2.38,23.08±1.31,25.16±1.53 respectively,Comparison between group A and group B existed in Significant difference(P＜0.001),Comparison between group C and group D existed in Significant difference(P＜0.01).
3.shrinkage rate of wound surface:The shrinkage rate of wound surface of four groups is 36.89±1.94%,39.56±2.93%,92.51±1.44%,92.40±1.91%respectively,Comparison between group A and group B existed in Significant difference(P＜0.01);Comparison between group C and group D has no difference(P=0.903)
Part three experimental study on proliferation and differentiation of the bone mesenchymal stem cell on wound surface
1.Examination in situ hybridization:
In the microskin specimen sections of group A,many cells which nucleolus is brown can be seen among hair follicle.A few brown nucleolus cells were scattering in the dermis;A few brown nucleolus cells were scattering in the healing scar tissue specimen sections of group C;Few these cells could be seen among specimen sections of group B and group D.
2.Examination of immune fluorescence histochemistry:
1) Red fluorescence of labeled BrdU and green fluorescence of labelled Keratin can be seen among hair follicle and epidermis base layer in group A specimen sections,and existed in the seem position;Green fluorescence of labelled Keratin can be seen among hair follicle and epidermis base layer in group C specimen sections,but no red fluorescence of labelled BrdU can be seen in group C specimen sections; In group D specimen sections red fluorescence of labeled BrdU and green fluorescence of labelled Keratin can not be seen.
2) Red fluorescence of labelled BrdU and green fluorescence of labelled FⅧcan be seen in the dermis of group A and healed scar of group C specimen sections,and formed a structure like vascular,and existed in the seem position;In the dermis of group B and healed scar group D specimen sections green fluorescence of labelled FⅧcan be seen, red fluorescence of labelled BrdU cannot be seen.
3) Red fluorescence of labelled BrdU and green fluorescence of labelled Actin can be seen in the dermis of group A and healed scar group C specimen sections,and existed in the seem position;In the dermis of group B and healed scar of group D specimen sections green fluorescence of labelled Actin can be seen,red fluorescence of labelled BrdU cannot be seen.
4) Red fluorescence of labelled BrdU and green fluorescence of labelled Nestin can be seen scattering in the dermis of group A and healed scar group C specimen sections,and existed in the seem position; Green fluorescence of labelled Nestin can be seen in the dermis in group C specimen sections,but no red fluorescence of labelled BrdU can be seen in group C specimen sections;In group D specimen sections red fluorescence of labeled BrdU and green fluorescence of labelled Nestin can not be seen.
5) Red fluorescence of labeled BrdU and green fluorescence of labelled S-100 can be seen scattering in the dermis of group A and healed scar group C specimen sections,and existed in the seem position;Green fluorescence of labelled Nestin can be seen in the dermis in group C specimen sections,but no red fluorescence of labelled BrdU can be seen in group C specimen sections;In group D specimen sections red fluorescence of labeled BrdU and green fluorescence of labelled Nestin can not be seen.
[Conclusion]
1.Isolated BMSCs from rat bone marrow successfully.
2.The BMSCs we isolated have multi-potential differentiation ability, accord with characteristic of adult stem cell.
3.The BMSCs we transplanted into wound surface promoted the survival of microskin tissue particles and accelerated the healing of wound surface.
4.The BMSCs we transplanted into wound surface affected by the microenvironment coming from wound surface and microskin tissue, maybe differentiated into vascular endothelial cells,epidermal cell, myofibroblast,peripheral nerve tissue cell.
缺乏供皮区和创面愈合时间长是大面积烧伤创面修复的难题,愈合时间延长将使患者机体长期处于消耗状态,从而抵抗力下降、易于发生感染和多器官功能衰竭。自从发明微粒植皮技术后,利用少量的中厚皮片制成微粒皮颗粒,来消灭大面积的创面成为可能。但微粒皮植皮也有其缺点:1.生成的皮肤缺乏完整的真皮结构,较薄非常容易破溃,再次形成创面;2.在微粒皮愈合后创面易产生大量增生瘢痕,影响机体的外观和功能;3.微粒皮本身在创面成活率不确定,有时难以达到理想的效果。
干细胞技术的出现为解决创面修复提供了新的手段,干细胞具有强大的增殖能力,在一定的条件下,可以分化成各种功能细胞。根据发育的阶段,干细胞分为胚胎干细胞和成体干细胞。由于胚胎干细胞的研究受到伦理学方面的困扰,细胞来源受限,因而成体干细胞的研究便受到人们的高度重视。骨髓基质干细胞(Bone mesenchymal stem cell,BMSCs)是一种成体干细胞;BMSCs修复创面的机制目前认为:1.干细胞具有自我更新的能力和强大的分化潜能,能对创伤引起的微环境的改变,根据局部修复的需要分化成所需要的修复细胞并整合到局部组织中;2.在创面局部被激活的BMSCs能够扮演“微型生物反应器”的功能,分泌多种生长因子和细胞因子,调节局部的细胞的增殖分化,细胞基质的形成,血管的形成等过程;3.在机体受到损伤启动炎症反应过程的同时,BMSCs能够在早期迁徙到受损组织参与组织修复。
本课题紧贴临床实际需要,旨在探索应用干细胞技术解决大面积烧伤创面修复这一大难题的途径,依据BMSCs增殖分化的原理,将微粒皮组织与BMSCs一起应用于新鲜创面,运用细胞培养,免疫反应标记、分子生物学、组织病理学微观检测等手段,监测BMSCs增殖分化,以及微粒皮增生爬行的情况。目前国内外尚未开展相关课题的研究。所得数据将为以下方面提供依据:
1.BMSCs在新鲜创而扮演怎样的“微型生物反应器”的功能,其增殖、分化的方向以及形成细胞基质的情况是怎样。
2.在新鲜创面上,BMSCs能否与微粒皮组织黏附发生侵入、迁徙、增殖并分化,促进微粒皮组织的成活。
3.BMSCs与微粒皮组织能否促进创面愈合,提高创面愈合质量。
[方法]
第一部分F344雄性大鼠BMSCs的体外分离培养及鉴定
1.BMSCs分离、培养、传代:断颈处死10～12W F344雄性大鼠,从股骨、胫骨,肱骨中冲洗出骨髓,接种于培养瓶,48h后更换培养液,弃去未贴壁的细胞,每隔48h换液,接近融合的BMSCs按1:2比例传代培养,留取第3代备用。
2.细胞生长动力学:取3、5代细胞MTT法测细胞增殖活性并绘制细胞增殖曲线。
3.BMSCs的细胞周期检测:取传代生长良好的的第3代细胞,0.25%胰酶室温消化后制成细胞悬液,碘化丙啶染色,流式细胞仪检测。
4.流式细胞仪检测细胞表面标记:第3代细胞,0.25%胰酶室温消化后制成细胞悬液,取浓度约1×10~6/ml 1ml分别加入适量FITC标记CD29,CD34,CD44,CD90抗体避光染色,流式细胞仪进行检测。
5.成脂、成骨诱导分化鉴定:第3代细胞,加入成骨、成脂诱导剂进行诱导油红“0”、茜苏红染色鉴定。
第二部分骨髓基质干细胞与微粒皮组织移植创面的实验研究
1.将P5代细胞用5-BrdU 10μmol/L孵育48h标记。
2.取Wistar大鼠全厚皮,剪成4×4cm~2大小作异体皮用。
3.48只F344雌性大鼠随机分为四组:A组骨髓间充质细胞+微粒皮移植组;B组微粒皮移植组;C组BMSCs移植组;D组生理盐水注射组。在F344大鼠背部形成一4×4cm~2创面,留取1/4皮片剪成微粒皮,均匀涂抹在异体皮上,缝合固定创面。
4.于术后14d打开异体皮,观察创面愈合情况,统计创面愈合率,观察并记录创面愈合时间,统计创面收缩率。5.在创面愈合的当日,处死大鼠,留取标本,放入-80℃冰箱保存。
第三部分骨髓基质干细胞在创面增殖、分化情况的实验研究
1.原位杂交
冰冻切片行大鼠Sry基因标记,光学显微镜下检查。
2.免疫荧光组织化学染色
冰冻切片进行BrdU、FⅧ、Keratin、Actin、Nestin、S-100免疫荧光组织化学染色,荧光显微镜下观察。
[结果]
第一部分BMSCs的分离培养扩增及鉴定
1.全骨髓细胞悬液接种于培养瓶后,24h体外培养有少量贴壁细胞,大量的悬浮细胞,48h后弃去悬浮细胞后继续培养,在96h换液后见悬浮细胞减少,细胞呈克隆样生长,10d左右达到细胞融合,基本上没有悬浮细胞。
2.细胞周期:碘化丙啶染色,流式细胞仪检测结果显示:MSCs中的S+G_2+M细胞约占20.19%,而处于G_0/G_1期的细胞为79.81%,大多数为合成期细胞,只有少数的细胞处于活跃的增殖期。
3.BMSCs表面标记的鉴定:CD29细胞阳性率为79.15%,CD34细胞阳性率为9.10%,CD44细胞阳性率为89.47%,CD90细胞阳性率为72.34%。
4.BMSCs成脂、成骨分化潜能的鉴定:成脂诱导14d油红“0”染色,细胞内可见细小脂滴形成,21d脂滴融合成片;成骨诱导14d茜苏红染色,可见少量钙盐沉淀,21d细胞间可见致密的、圆形、成片状的棕红团块,面积较大。
第二部分骨髓基质干细胞与微粒皮组织移植创面的实验研究
1.创面愈合率:伤后14d打开异体皮,A组创面已基本愈合,创面愈合率为85.77±3.47%;B组创面愈合率为62.16±4.39%;C组、D组创面为肉芽创面,创面愈合率分别为30.41±1.73%,29.96±1.33%,创面的愈合是因为创面的收缩。Welch=1028.700,P＜0.001,A、B两组比较有显著性差异P＜0.001,C、D两组比较没有显著性差异,P=0.898。
2.创面愈合时间:A组的创面愈合时间为17.17±1.64;B组为20.33±2.38;C组为23.08±1.31;D组为25.16±1.53。F=46.372,P＜0.001,A、B两组比较有显著性差异P＜0.001,C、D两组比较有显著性差异,P=0.006。
3.创面收缩率:A组的创面收缩率为36.89±1.94%;B组为39.56±2.93%;C组为92.15±1.44%;D组为92.40±1.91%;F=2608.977,P＜0.001,A、B两组比较有显著性差异P＜0.01,C、D两组比较没有显著性差异,P=0.903。
第三部分骨髓基质干细胞在创面增殖、分化情况的实验研究
1.原位杂交:A组微粒皮标本切片上,在毛囊的周围可见较多的,细胞核为棕色的细胞,真皮层内可见散在的核为棕色的细胞。C组标本在愈合瘢痕组织内可见散在的核为棕色的细胞。B组、D组标本未见核为棕色的细胞。
2.免疫组化双荧光染色:
1) A组标本可见较强的标记BrdU的红色荧光和Keratin的绿色荧光出现在毛囊周围,并且位于同一位置;B组标本可见较强的绿色荧光位于上皮基底层,但未见到相应BrdU标记的红色荧光。D组标本未见两种颜色的荧光。
2) A组与C组在真皮层内可见血管样结构的标记BrdU的红色荧光和FⅧ的绿色荧光位于同一位置;B组标本在真皮层内可见散在的绿色荧光,D组标本在愈合瘢痕组织可见散在的绿色荧光,但两组均未见相应的标记BrdU的红色荧光出现。
3) A、C两组在真皮层内可见散在的标记BrdU的红色荧光和Actin的绿色荧光位于同一位置;B组标本在真皮层内可见散在的绿色荧光,D组标本在愈合瘢痕组织可见散在的绿色荧光,但两组均未见相应的标记BrdU的红色荧光出现。
4) A、C两组在真皮层内可见散在的标记BrdU的红色荧光和Nestin的绿色荧光位于同一位置。B、D组标本未见两种颜色的荧光。
5) A、C两组在真皮层内可见散在的标记BrdU的红色荧光和Nestin的绿色荧光位于同一位置。B、D组标本未见两种颜色的荧光。
[结论]
1.成功地从大鼠骨髓中分离出BMSCs,并证实分离的BMSCs具有多项分化潜能,符合成体干细胞的特性
2.移植创面的BMSCs能促进微粒皮组织成活,并促进创面愈合
3.移植于创面的BMSCs在创面及微粒皮组织微环境的作用下,根据创面修复的需要,可能分化成为血管内皮细胞、肌成纤维细胞、表皮细胞,周围神经组织细胞。
[Background and Objective]
The two difficult problems of severe burns are Lack of skin donor sites and delayed time of skin wound healing,delayed time of skin wound healing can result in a consuming state of body in a long period.This can lead to the descent of resistance abilities of body,easy getting infections and multiple organ failure.Since the invention of microskin transplantation technique,utilizing a small quantity of splitthickness skin graft to make into miroskin tissue particles,the extinction of wound surface be become into reality.This bring a revolutionary breakthrough in repairing severe burns wound surface. There are some shortcomings of microskin transplantation in this technique:1.The skin by microskin extended is lack of integral dermis, very thin and easy to ulcerate,be prone to become wound surface again; 2.After the wound surface healing of applying microskin tissue particles transplantation,a mass of hyperplasia scar come into being, affected the appearance and function of the body.3.The surviving of microskin on wound surface is not insure some time,and will not get a good result.
The coming forth of stem cell technique bring a new way in repairing the wound surface.The stem cell can be provided with strong hyperplastic ability.In a certain condition,it can be differentiated into kinds of functional cells.Base on the phase of cell development,stem cell be divided into two categories:embryonic stem cell and adult stem cell. The research in embryonic stem cells is restricted by its limited source and ethical controversy,so research on adult stem cells attracts more attentions.Bone marrow mesenchymal stem cell is a kind of adult stem cell,derived from mesoderm.The mechanism of BMSCs repairing wound surface lie in the following ways:1.Stem cells have self-regenerate ability and strong differentiate ability.Adapt to the microenvironment change on wound,it can be differentiated into kinds of cells and integrated into local tissues according to the needs of the body.2.The enabled BMSCs in wound surface can act as a micro-bioreactor,it can secrete kinds of growth factor and cell factor,accommodate the proliferation and differentiation of local tissue,the formation of cell matrix,the formation of vascular et al;3.Once the body has been damaged, at the same time of starting process of inflammation reaction,BMSCs can migrate into damaged tissue and join the repairing of the tissue.
This project bases on the principle of the proliferation and differentiation of BMSCs.Transplanted the BMSCs and microskin tissue into fresh wound surface,utilize cell culture immunological marking and reaction,molecular biology histopathology microcosmic detection, monitoring the proliferation and differentiation of BMSCs and the hyperplasia and extending of microskin particles,The experimental data will provide the evidences for the following way:
1.How are the BMSCs acted as micro-bioreactor in fresh wound surface, and how are the orientation of proliferation and differentiation and the way of forming cell matrix.
2.Whether BMSCs can attach to and invade into and migrate into microskin tissue particles,proliferating and differentiating,and improve the surviving of microskin tissue particles in fresh wound surface.
3.Whether the BMSCs and microskin tissue particles can accelerate the healing of wound surface,and improve the healing quality of wound surface.
[Methods]
Part one Isolation and identification of BMSCs of male F344 rat in vitro
1.Isolation,culture and passage:break off the neck and execute male F344 rat,douche the bone marrow from femur,tibia and humerus, inoculate these cells into culture flask,replace the culture media after 48 hours,remove the non-adherent cells.Culture media was changed every 48 hours.Cells were passaged with ratio of 1:2 when almost confluent.Passage 3 cells were used for further experiment.
2.Cell growth kinetics:cell proliferation activity of passage 3 and passage 5 cells were measured,cell proliferation curve were be drawn.
3.Cell cycle examination of BMSCs:active growing passage 3 cells were detached by 0.25%trypsin and made into single cell suspension,stained for propidium iodide,then passed through flow cytometer.
4.Cell surface marker examination of BMSCs by flow cytometer:active growing passage 3 cells were detached by 0.25%trypsin and made into single cell suspension,take 1ml cells which concentration is 1×10~6/ml and stain for CD29,CD34,CD44 and CD90 labelled with FITC,then measured by flow cytometer.
5.Lipogenesis and osteogenesis induction and characterization: passaged 3 cells were induced with lipogenetic reagent and osteogenetic reagent,stained for Oil Red 0 and Alizarin Red S, measured by microscope.
Part two experimental study on transplantation of bone mesenchymal stem cell and microskin tissue into wound surface
1.take passage 5 cells labeled with 5-Brdu(10μmol/L) for 48 hours.
2.Take full thickness skin graft of Wistar rat,cut into size 4×4cm~2
3.48 F344 female rat take into four groups:group A transplant bone mesenchymal stem cell and microskin tissue particles into wound surface;group B transplant microskin tissue particles into wound surface;group C transplant bone mesenchymal stem cell into wound surface;group D injected with physiological saline solution.Forming a wound surface of size 4×4cm~2,take a quarter of full thickness skin graft cut into microskin tissue particles,lay on surface of xenoma skin,suture and fix on the surface.
4.Opened up the xenoma skin 14 days post-operation,observed and record healing rate of wound surface;Observed and recorded healing time of wound surface;Record the shrinkage rate of wound surface.
5.On the day of wound surface healing,execute the rat,took the skin specimen,preserved the specimen on refrigerator of -80℃.
Part three experimental study on proliferation and differentiation of the bone mesenchymal stem cell on wound surface
1.hybridization in situ
Rat sry gene was labelled in freezing section,measured in optical microscope.
2.Examination of immune fluorescence histochemistry
Freezing sections were stained for Cy3 conjugated secondary antibody combined with BrdU antibody,FITC conjugated secondary antibody combined with FⅧ,Keratin,actin,nestin and S-100 antibody,analysed with fluorescence microscope.
[Result]
Part one Isolation and identification of BMSCs of male F344 rat in vitro
1.After the full bone marrow cell suspension was inoculated in a culture flask,a small amount of bone marrow mononuclear cells adhere to the flask 24 hours after planting.After 48 hours,suspending cells were discarded.In 96 hours,suspending cells were decreased obviously,the cells were be growing with clone formation,reaching homogeneous confluence in 12 hours.In 10 days,the cells were fusion,cannot see the suspending cells basically.
2.Flow cytometer analysis showed that about 20.19%of cells are in S+G2+M phase,79.81%are in Go/G phase,and only small numbers are in active proliferation state.
3.Identification of BMSCs superficial marker:79.15%cells were CD29 positive cells;9.10%cells were CD34 positive cells;89.47%cells were CD44 positive cells;72.34%cells were CD90 positive cells.
4.lipogenesis and osteogenesis induction and characterization:After 14 days lipogenesis induction,and stained for Oil Red O,many tiny lipid droplet can be seen in cytoplasm,In 21 days,lipid droplet in cytoplasm fused,like vacuole,osteogenesis induction stained for Alizarin Red S,in 14 days,calcium salt deposition can be seen;In 21 days,brown red mass can be seen among the induction cells.
Part two experimental study on transplantation of bone mesenchymal stem cell and microskin tissue particles into wound surface
1.Healing rate of wound surface:xenoma skin was opened in 14 days,wound surface of group A had been healed basically,healing rate of wound surface was 85.77±3.47%;healing rate of wound surface was 62.16±4.39%;The wound surface of group C,group D was flesh bud tissue healing rate of wound surface was 30.44±1.73%,29.96±1.33% respectively;The healing of wound surface was own to shrinkage of wound surface.Comparison between group A and group B existed in Significant difference(P＜0.001);Welch=1028.700,P＜0.001;Comparison between group C and group D has no difference(P=0.898)
2.Healing time of wound surface:The healing time of wound surface of four groups is 17.17±1.64,20.33±2.38,23.08±1.31,25.16±1.53 respectively,Comparison between group A and group B existed in Significant difference(P＜0.001),Comparison between group C and group D existed in Significant difference(P＜0.01).
3.shrinkage rate of wound surface:The shrinkage rate of wound surface of four groups is 36.89±1.94%,39.56±2.93%,92.51±1.44%,92.40±1.91%respectively,Comparison between group A and group B existed in Significant difference(P＜0.01);Comparison between group C and group D has no difference(P=0.903)
Part three experimental study on proliferation and differentiation of the bone mesenchymal stem cell on wound surface
1.Examination in situ hybridization:
In the microskin specimen sections of group A,many cells which nucleolus is brown can be seen among hair follicle.A few brown nucleolus cells were scattering in the dermis;A few brown nucleolus cells were scattering in the healing scar tissue specimen sections of group C;Few these cells could be seen among specimen sections of group B and group D.
2.Examination of immune fluorescence histochemistry:
1) Red fluorescence of labeled BrdU and green fluorescence of labelled Keratin can be seen among hair follicle and epidermis base layer in group A specimen sections,and existed in the seem position;Green fluorescence of labelled Keratin can be seen among hair follicle and epidermis base layer in group C specimen sections,but no red fluorescence of labelled BrdU can be seen in group C specimen sections; In group D specimen sections red fluorescence of labeled BrdU and green fluorescence of labelled Keratin can not be seen.
2) Red fluorescence of labelled BrdU and green fluorescence of labelled FⅧcan be seen in the dermis of group A and healed scar of group C specimen sections,and formed a structure like vascular,and existed in the seem position;In the dermis of group B and healed scar group D specimen sections green fluorescence of labelled FⅧcan be seen, red fluorescence of labelled BrdU cannot be seen.
3) Red fluorescence of labelled BrdU and green fluorescence of labelled Actin can be seen in the dermis of group A and healed scar group C specimen sections,and existed in the seem position;In the dermis of group B and healed scar of group D specimen sections green fluorescence of labelled Actin can be seen,red fluorescence of labelled BrdU cannot be seen.
4) Red fluorescence of labelled BrdU and green fluorescence of labelled Nestin can be seen scattering in the dermis of group A and healed scar group C specimen sections,and existed in the seem position; Green fluorescence of labelled Nestin can be seen in the dermis in group C specimen sections,but no red fluorescence of labelled BrdU can be seen in group C specimen sections;In group D specimen sections red fluorescence of labeled BrdU and green fluorescence of labelled Nestin can not be seen.
5) Red fluorescence of labeled BrdU and green fluorescence of labelled S-100 can be seen scattering in the dermis of group A and healed scar group C specimen sections,and existed in the seem position;Green fluorescence of labelled Nestin can be seen in the dermis in group C specimen sections,but no red fluorescence of labelled BrdU can be seen in group C specimen sections;In group D specimen sections red fluorescence of labeled BrdU and green fluorescence of labelled Nestin can not be seen.
[Conclusion]
1.Isolated BMSCs from rat bone marrow successfully.
2.The BMSCs we isolated have multi-potential differentiation ability, accord with characteristic of adult stem cell.
3.The BMSCs we transplanted into wound surface promoted the survival of microskin tissue particles and accelerated the healing of wound surface.
4.The BMSCs we transplanted into wound surface affected by the microenvironment coming from wound surface and microskin tissue, maybe differentiated into vascular endothelial cells,epidermal cell, myofibroblast,peripheral nerve tissue cell.
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