摘要
一、背景
近年来原林教授等提出筋膜学假说认为：人体由非特异性结缔组织筋膜支架所构成的支持与储备系统和被筋膜支架所包绕的功能系统所共同构成。支持与储备系统的筋膜支架是以干细胞为核心,为功能系统的更新提供细胞补充,并为功能系统的各种细胞的更新、代谢提供一个稳定的内部环境。脂肪组织是支持与储备系统的重要组成部分,其中的脂肪间充质干细胞(adipose-derived mesenchymal stem cells, ADMSCs)是机体重要的干细胞储备之一。对机体不同部位(如内脏和皮下脂肪组织)中的ADMSCs深入研究,分析其免疫表型的差异以及多向分化能力有无差别,能对筋膜学假说提供部分理论支持,同时也是进行干细胞移植取材时应该考虑的问题。
ADMSCs也是目前干细胞的研究热点之一。间充质干细胞具备成体干细胞的特征,能无限传代和多向分化。目前已经从多种组织中分离出间充质干细胞,包括骨髓、脂肪、脐带血、外周血和骨骼肌等。间充质干细胞中研究较为深入的是骨髓间充质干细胞,能在一定的条件下分化为骨细胞、脂肪细胞、软骨细胞、骨骼肌细胞和神经细胞等,骨髓间充质干细胞是首先分离的间充质干细胞,已经作为种子细胞在各种移植实验中取得了明显的效果。近期的研究发现,同样具有多向分化能力的ADMSCs和骨髓间充质干细胞相比具有取材手术小、可重复进行、酶消化分离程序简单等优点,可作为新的成体干细胞来源,引起了许多研究者的兴趣。
自体ADMSCs作为种子细胞是理想的,为获取大量的自体ADMSCs作为种子细胞,需要获得脂肪组织后在体外进行分离扩增。机体作为获取自体ADMSCs的脂肪组织供体,一般处于某种病理状态下(如创伤状态下)。在这种病理状态下能否获得足够数量和质量的自体ADMSCs,机体的病理状态是否对ADMSCs的多向分化和表面标志造成了影响,是进行ADMSCs移植治疗研究中应该考虑的问题之一。
创伤性脑损伤的治疗是目前世界范围内的一个难题。脑损伤后病理过程复杂,而且神经系统本身的再生能力差,在治疗方面面临许多难题。但目前的各种治疗方法都不能达到理想的效果,近年来干细胞移植治疗给创伤性脑损伤的治疗带来了希望,许多学者进行了大量的实验研究。细胞移植治疗脑损伤的机理可能是促进了神经营养因子的分泌,或者是替代了损伤的神经细胞。我们研究的内容是静脉移植ADMSCs能否促进大鼠创伤性脑损伤的恢复以及对脑源性神经营养因子和胶质细胞源性营养因子的影响。
二、目的
1、分离培养SD大鼠的ADMSCs,并进行表面标志CD11b、CD29、CD45、CD49d、CD90和CD106的检测,进行成骨成脂诱导检测多向分化能力；
2、内脏(大网膜)和皮下取材获得的ADMSCs在成骨成脂分化能力定量测定、增殖能力以及表面标志是否存在差异；
3、创伤状态(皮肤切口)对获取的ADMSCs的收获率、增殖能力和表面标志是否造成了影响；
4、静脉移植ADMSCs能否促进创伤性脑损伤大鼠的神经功能恢复,能否迁移到损伤区域以及对脑源性神经营养因子和胶质细胞源性神经营养因子含量有无影响
三、方法
1、ADMSCs的分离培养、多向分化、表面标志鉴定
从6只SD大鼠腹股沟脂肪垫中分离培养ADMSCs。先将脂肪组织剪碎后用Ⅰ型胶原酶消化,离心将上层脂肪细胞从间质血管碎片中去除,然后以8000-10000cells/cm2的密度种植于培养瓶中。培养基为含10%胎牛血清和1%青霉素/链霉素的DMEM。24h后换液去掉未贴壁细胞,贴壁细胞用来扩增传代。相差显微镜下观察ADMSCs的形态。
取第4代ADMSCs进行成骨和成脂诱导分化,经成骨诱导培养基诱导2周后用茜素红染色检测矿化结节。经成脂诱导培养基诱导2周后用油红O染色检测脂质沉积。
用流式细胞术鉴定第4代的ADMSCs的表面标志物：CD11b、CD29、CD45、CD49d、CD90和CD106的表达率。
2、内脏(大网膜)和皮下ADMSCs的比较
6只SD大鼠取大网膜和腹股沟脂肪垫,培养扩增ADMSCs。相差显微镜下观察两个部位来源的ADMSCs的形态。分别取两种来源的第4代ADMSCs进行成骨和成脂诱导分化。每只大鼠来源的第4代腹股沟和大网膜ADSCs分别种植于6孔板,成脂定量方法：成脂诱导后用油红O染色检测细胞内脂质沉积,后用异丙醇洗脱,在510nm波长测定光密度值OD。成骨定量方法：ADMSCs成骨诱导后用茜素红染色用10%西吡氯铵洗脱,在560nm波长测量光密度值OD。用3个6孔板平均每孔的OD值来表示每只大鼠来源的ADMSCs的成骨成脂分化能力
取两种第4代ADMSCs,计算细胞数目后种植于24孔培养板,每个24小时消化3个孔后计算细胞数。每孔计数2次,3孔的平均值为该时间点细胞数。绘制细胞生长曲线,根据公式TD=t×lg2/(lgNt-lgNo)计算出各组ADMSCs倍增时间(TD)。其中t为细胞对数生长时间,Nt为对数生长期终止时间的细胞数,N0为细胞生长曲线的对数生长期起始时间点细胞数,计算细胞倍增时间。流式细胞术检测两种来源的第4代ADMSCs表面标志CD11b、CD29、CD45、CD49d、CD90和CD106的表达率。
3、皮肤损伤对ADMSCs的影响
6只SD大鼠在左侧腹股沟做皮肤切口后缝合,7d后和6只健康鼠同取右侧腹股沟脂肪垫分离培养ADMSCs。计算对照组和皮肤切口组每mg脂肪组织收获到的间质血管碎片细胞数和贴壁细胞。
用这两种第4代ADMSCs进行成骨成脂诱导分化,绘制生长曲线并测定倍增时间。流式细胞术检测表面标志CD11b、CD29、CD45、CD49d、CD90和CD106的表达率。
4、静脉移植ADMSCs对创伤性脑损伤的影响
创伤性脑损伤模型的建立采用大鼠脑冷冻伤模型作为创伤性脑损伤模型。36只体重200-220克SD大鼠随机分成3组,实验组、对照组、正常组各12只。实验组和对照组动物水合氯醛腹腔注射麻醉成功后,手术显微镜于中线切开头皮,剥离骨膜,在左侧颞顶叶皮层上用牙科磨钻磨出一直径为5mm的骨窗,保持硬脑膜完整。将-80℃预冷直径4mm重10g的铜棒与硬脑膜充分接触1分钟后缝合头皮。正常组不给任何处理。
静脉移植脂肪间充质干细胞：SD大鼠ADMSCs扩增至第四代,用Brdu标记。用于移植的第4代ADMSCs在进行第4代传代后培养基中加入10μmol/L的Brdu,培养3天后消化收集,用生理盐水重悬,300万/ml。实验组大鼠创伤性脑损伤24h后行ADMSCs尾静脉移植,每只大鼠经尾静脉注射间充质干细胞3×106个。
Morris迷宫检测认知功能的恢复：从大鼠脑损伤后第11天至第14天行Morris迷宫检测,每天检测时间为上午9点至下午5点,记录找到逃生平台的时间(潜伏期)。检测4天后,移动并暴露逃生平台,记录到达逃生平台时间。
Brdu免疫荧光检测移植的ADMSCs在脑损伤区域的分布：从3组大鼠中每组取3只经心脏灌注4%多聚甲醛后断头取脑,常规制成石蜡切片。以损伤区域为中心切成5μm切片,行Brdu免疫荧光染色,了解移植的ADMSCs在脑内的分布情况。
BDNF和GDNF的定量检测：每组9只剩余大鼠立即断头处死取脑,放入液氮中,行western-blot检测BDNF和GDNF的相对含量。Kodak Image Station2000MM成像系统采集图像,获得图像用图像处理软件Image Tool 3.0处理,用BDNF/Actin代表BDNF的相对表达量.用GDNF/Actin代表GDNF的相对表达量。
四、结果
1、SD大鼠ADMSCs呈现类似成纤维细胞样形态学特征。1到4代ADMSCs在倒置显微镜下观察,经过传代后细胞形态逐渐均一,类似成纤维细胞,多呈长梭型或者纺锤形。用第4代ADMSCs进行成骨和成脂诱导分化,经诱导后能向成骨细胞和脂肪细胞转化。成脂培养基内诱导两周后,部分内脂滴积聚。细胞在培养皿上展开,脂滴占据细胞大部,胞核被挤于一侧。油红O染色显示特异性的红色脂滴。成骨诱导后细胞逐渐聚集,并可重叠生长,两周后可见细胞外大量钙质沉积,矿化结节被茜素红染成红色的致密结节。免疫表型分析显示CD29和CD90呈阳性,而CD11b、CD45、CD49d和CD106呈阴性反应。
2、大网膜和腹股沟来源的ADMSCs在细胞形态上没有显著区别,原代细胞和各代细胞在倒置显微镜下观察,细胞多呈长梭型,间有不规则型和三角形。两个部位来源的ADMSCs均能进行成脂和成骨诱导分化。在进行成骨和成脂诱导后,分别经西吡氯铵和异丙醇洗脱后测定OD值,结果两者分化能力无明显差异。
腹股沟脂肪垫和大网膜来源的第4代ADMSCs生长曲线形态接近。根据生长曲线计算出细胞倍增时间分别为0.72±0.05天和0.73±0.06天,两组比较P>0.05,无统计学意义。
两个部位来源的ADMSCs中CD29和CD90均呈高表达,CD11b、CD45、CD49d和CD106表达率呈低表达。CD11b、CD29、CD106和CD90的表达率无明显差异,P>0.05,CD45和CD49d的表达率有显著差异,P<0.05。
3、两组大鼠获得的ADMSCs原代细胞及传代细胞形态无明显差别。皮肤切口组大鼠间质血管碎片细胞的收获率(0.47±0.20×104/mg)明显低于对照组大鼠的收获率(1.43±0.17×104/mg),贴壁细胞收获率(1.29±0.52×104/mg)也明显低于对照组(3.84±0.54×104/mg),均为P<0.05,差异具有统计学意义。
从对照组和皮肤切口组大鼠得到的第4代ADMSCs均能进行成骨诱导分化。
对照组和皮肤切口组第4代ADMSCs生长曲线形态接近。对照组和皮肤切口组收获的第4代ADMSCs倍增时间分别为1.11±0.06d和1.09±0.05d,两组比较p>0.05,无统计学意义。
对照组和皮肤切口组第4代ADMSCs表面标志中CD106、CD49d、CD45、CD11b均呈低表达,CD29和CD90呈高表达,两组比较p>0.05,无统计学意义。
4、ADMSCs移植明显促进了创伤性脑损伤大鼠的认知功能恢复
Morris迷宫检测结果：3组大鼠均完成Morris迷宫检测,没有出现体重下降等健康问题。实验分组对大鼠的潜伏期的影响差异明显,P<0.05,实验组大鼠的测量4天中每天的潜伏期均低于对照组,迷宫检测第5天到达逃生平台的时间无差异。
免疫荧光检测显示移植的ADMSCs大量聚集在损伤的大脑皮层,在正常皮层和其他区域未见有移植的细胞迁移。
Western-blot检测显示移植组大鼠脑组织中BDNF和GDNF的相对表达量明显高于单纯损伤组,差异显著,P=0.000。
五、结论
1、通过这种酶消化方法获得的SD大鼠ADMSCs具有成骨成脂多向分化能力,免疫表型分析显示符合间充质干细胞的特征。
2、SD大鼠大网膜和腹股沟脂肪垫来源的ADMSCs均能成骨成脂分化,成骨成脂分化能力定量测定无明显差别,免疫表型不完全相同。
3、皮肤切口能引起皮下脂肪组织的ADMSCs收获率降低,对获得的ADMSCs在形态、多向分化性质、增殖能力和表面标志方面无明显影响。
4、静脉移植ADMSCs能定植于创伤性脑损伤大鼠的损伤皮层,接受移植大鼠脑内BDNF和GDNF的含量增加,神经功能明显改善。
Background
Recently, Lin Yuan has put forward a new hypothesis, fasciaology. In fasciaology, the human body is classified into two major systems. One is the supporting-storing system, which is consisted of undifferentiated cells of unspecialized connective tissues. The other is the functional system, which is consisted of differentiated functional cells and is enclosed by the supporting-storing system. The undifferentiated stem cells in the supporting-storing system incessantly differentiate into functional cells. The supporting-storing system throughout the body regulates the functional and living status of differentiated cells and provides a stable internal environment for the survival of these cells. Adipose tissue is a an important part of the supporting-storing system, and adipose-derived mesenchymal stem cells (ADMSCs) are main stem cells reserved in this system. We investigated the difference of stem cells reserved in different site, for example, in greater omentums and subcutaneous adipose tissues, by comparing the characteristics of multilineage differentiation potential and cell surface markers of ADMSCs. This research will contribute to fasciaology and cell therapy.
In recent years, interest has rapid grown in the research of ADMSCs. Being one kind of mesenchymal stem cells, ADMSCs have the capacity to selfrenew for indefinite periods and can differentiate to many different cell types. Mesenchymal stem cells can be isolated from several organs, such as bone marrow, fat, umbilical cord blood, peripheral blood and skeletal muscles. A significant number of investigations have focused on mesenchymal stem cells derived from bone marrow, which can differentiate to multiple cell phenotype, including bone, fat, cartilage, skeletal muscle and neural progenitor, under appropriate culturing conditions. Bone marrow mesenchymal stem cells were first identified and are one of the most widely used stem cell sources. Therapeutic potential of transplantation of them is invigorating. Compared with bone marrow mesenchymal stem cells, ADMSCs do have an equal potential to differentiate into multiple cell phenotype. However, the simple surgical procedure, the easy and repeatable access to the subcutaneous adipose tissue, and the uncomplicated enzyme-based isolation procedures make ADMSCs most attractive for researchers. Being a new source of therapeutic stem cells, ADMSCs should be given more attention to.
Autologous ADMSCs as seed cells are ideal for autologous stem cell transplantation. In order to obtain a large number of autologous seeded cells, ADMSCs should be idolated from adipose tissue and cultured in vitro. As the ADMSCs donor, organisms are often in pathological situation (such as trauma). It should be investigate that whether there are sufficient ADMSCs in such situation and the influence of trauma on the multiple differentiations and cell surface markers.
Traumatic brain injury (TBI) is a major health problem in the world. The therapy of TBI faces many difficulties as a result of the limited ability of the central nervous system for self-renewal and complicated pathological processes present in the state of the brain disorder. Currently, there is no effective clinical treatment to promote recovery after TBI. However, a new interventions, cellular therapy, is being explored in experimental studies. Considerable hope is vested in cellular therapy, in which transplanted cells would be the source neurotrophic factors and could also be the source of new cells. We investigated if intravenous transplatation of ADMSCs results in functional recovery after TBI in rats and the influence of cell transplatation on brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) release.
Objective
1. To isolate and cultivate ADMSCs from SD rat and conduct immunophenotypic characterization with the following cell surface markers:CDllb, CD29, CD45, CD49d, CD90 and CD 106. And to induce adipogenic and osteogenic differentiation of ADMSCs and conform their differentiation potential.
2. To investigate the difference of the immunophenotypic characterization by comparing the cell surface markers profile and the proliferative capacity of ADMSCs reserved in viscera (greater omentums) and subcutaneous adipose tissues in Sprague-Dawley rats. And to compare the quantitative osteogenesis assay and the quantitative adipogenesis assay.
3. To investigate the influence of skin wound on the yield, growth characteristics and cell surface markers of ADMSCs in rats.
4. To investigate the effect of intravenous transplatation of ADMSCs on functional recovery after TBI in rats, the migration of ADMSCs in the injured brain and the influence of cell transplantation on BDNF and GDNF.
Methods
1. The isolation and culture of ADMSCs of SD rats, multi potentiality and the characterization of cell surface markers:
ADMSCs of 6 Sprague-Dawley rats from inguinal fat pads were isolated and cultured. Briefly, the adipose tissues were mechanically dissociated and digested with collagenase type I. The adipocytes were separated from the stromal vascular fraction by centrifuging the suspension. The cells in the stromal vascular fraction were plated in flasks at a density of 8000-10,000 cells/cm2. The cells were cultured with Dulbecco's Minimal Essential Medium (DMEM) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. After 24 hours, the non-adherent cells were removed and the adherent cells were expanded by serial passage. The morphological characterization of ADMSCs was observed using phase-contrast microscopy.
ADMSCs at passage 4 were cultured under adipogenic and osteogenic condition to conform their differentiation potential. The morphological characterization of inductive cells was observed using histological staining such as alizarin red for mineralization nodules and oil red O for lipid accumulation.
The immunophenotypic marker including CD11b, CD29, CD45, CD49d, CD90 and CD106 of ADMSCs grown for 4 passages was determined using flow cytometry.
2. The comparison of ADMSCs from viscera (greater omentum) and subcutane ous:
ADMSCs were isolated and cultured from greater omentums and inguinal fat pads of 6 Sprague-Dawley rats. The morphological characterization of ADMSCs was observed using phase-contrast microscopy. ADMSCs at passage 4 were cultured under adipogenic and osteogenic condition to conform their differentiation potential. The morphological characterization of inductive cells was observed using histological staining such as alizarin red for mineralization nodules and oil red O for lipid accumulation. ADMSCs from two sites were plated in 6-well plates. Adipogenic differentiation was induced by culturing ADMSCs in adipogenic medium and assessed using an Oil Red O stain as an indicator of intracellular lipid accumulation. For the quantitative adipogenesis assay the cells were de-stained with isopropyl alchohol. The amount of Oil Red O was determined by measuring the optical density of the solution at 510 nm. Osteogenesis differentiation was induced by culturing ADMSCs in osteogenesis medium and examined for extracellular matrix calcification by Alizarin Red staining. For the quantitative osteogenesis assay, the cells wer de-stained with 10% cetylpyridinium chlorid. The amount of Alizarin Red was determined by measuring the optical density of the solution at 560 nm. The average optical density of wells represents the quantitation of the levels of differentiation in ADMSCs.
The ADMSCs at passage 4 from two sites were seeded in 24-well plates. Every 24 hours after seeding, cells from 3 wells were harvested and cells in each well were counted twice. Average ADMSCs numbers were plotted against the number of days cultured. The growth curves were investigated and the logarithmic growing phase of the cells was determined. The population doubling time was calculated using the formula:(days in logarithmic phase)/((logN2—logNl)/log2), where N1 is the number of cells at the beginning of the logarithmic growing phase and N2 the number of cells at the end of the logarithmic growing phase.
The phenotypical marker profile including CD11b, CD45 and CD90 of ADMSCs from two sites grown for 4 passages was determined using flow cytometry.
3. The influence of skin wound on adipose-derived mesenchymal stem cells harvested from subcutaneous adipose tissue in rats:
6 Sprague-Dawley rats were suffered skin incision in left inguen and the wounds were sutured. After 7 days, ADMSCs of injured rats and 6 normal rats from right inguinal fat pads were isolated and cultured. The yield of stromal vascular fraction cells and adherent cells were investigated. Both kind of ADMSCs at passage 4 were induced to adipogenic and osteogenic differentiation. Growth kinetics were investigated and doubling times were determined. The phenotypical marker profile including CD11b, CD29, CD45, CD49d, CD90 and CD106 of ADMSCs grown for 4 passages was determined using flow cytometry.
4. The influence of intravenous transplantation of ADMSCs on functional recovery after traumatic brain injury in rats:
Traumatic brain injury model:
Experimental TBI was induced in rats by a cryogenic injury model.36 SD rats (weighting 200 to 220 g) were randomly divided into three groups, the experimental group (n=12), the control group (n=12) and the normal group (n=12). The experimental group and control group animals were anesthetized with chloral hydrate intraperitoneally. Under surgical microscope the skull was exposed by a midline incision and a circular hole (approximately 5 mm in diameter) was drilled on the skull over the left temporoparietal cortex leaving the dura intact by using a dental drill. The precooled copper probe (-80℃) 4 mm in diameter with 10-g weight was lowered onto the dural surface and kept in place for 60 s. The skin was sutured. The rats in normal group were not injured.
Intravenous transplantation of ADMSCs:
ADMSCs of SD rat were isolated and cultured. ADMSCs at passage 4 were harvested after proliferating in the medium added 5-bromo-2-deoxyuridine (BrdU) (10μmol/L). One day after TBI, the rats in the experimental group were injected with ADMSCs labled with BrdU via the tail vein(3×106 cells in 1ml saline/each).
Determination of cognitive function:
Morris water maze tests were performed from day 11 to 14 after TBI between 9:00 and 17:00. The time to reach the platform (latency) was measured. The day after the acquisition phase (day 4), a test was conducted by moving and revealing the platform. The time reached the target, the visible platform, from the opposite quadrant was recorded.
Immunofluorescent staining:
Three rats in every group were anesthetized after Morris water maze tests. They next received intracardiac perfusion of 4% paraformaldehyde in PBS. Rat brains were postfixed overnight and then routine paraffin sections were made. Serial transverse sections at a thickness of 5μm were cut through the injured district. The expressions of the incorporation of BrdU were investigated using immunofluorescent staining and the migration of transplanted ADMSCs were determined.
Quantitative analysis of BDNF and GDNF:
The other 9 rats in each group were sacrificed by rapid decapitation on day 15. The brains were quickly removed and stored in liquid nitrogen until use. Western blott was used for measuring levels of BDNF and GDNF. The images were captured by Kodak Image Station 2000 MM system and data were processed using image processing software Image Tool 3.0. The levels of BDNF and GDNF were expressed as the ratio of studied protein versus actin.
Results
1. Primary culture cells showed fibroblastic-like morphologic characteristics. Within one to four passages, ADMSCs appeared as a monolayer of large, flat cells, and became relatively homogeneous in appearance as the cells were passaged. ADMSCs had the ability to differentiate along adipogenic and osteogenic lineages. When cultured under adipogenic condition for 2 weeks they were induced toward the adipogenic lineage. A fraction of the cells contained multiple, intracellular lipid-filled droplets that accumulated Oil Red-O. The Oil Red O-containing ADMSCs exhibited an expanded morphology with the majority of the intracellular volume occupied by lipid droplets, consistent with the phenotype of mature adipocytes. When cultured ADMSCs were exposed to an osteogenic induction medium, they aggregated and formed calcium deposits after 2 weeks. An alizarin red stain for precipitated calcium salt was performed on differentiated cells. Calcification appears as red regions within the cell monolayer. The immunophenotypes analysis revealed that the rat ADMSCs were positive for CD29 and CD90, but negative for CDllb, CD45, CD49d and CD 106.
2. The morphological characterization of ADMSCs harvested from greater omentums and inguinal fat pads were almost the same. Primary culture cells and cells within one to four passages from two sites showed the same fibroblastic-like morphologic characteristics. Both of them have the capacity to differentiate toward the adipogenic and osteogenic lineages. The average optical density representing the quantitation of the levels of differentiation was confirmed after ADMSCs were induced in adipogenic and osteogenesis differentiations. The abilities to differentiate along adipogenic and osteogenic lineages showed no significant difference between two kinds of cells. The growth curves of cultured ADMSCs at passage 4 from the two sites showed no obvious difference. The population doubling times based on the growth curves were similar between ADMSCs from greater omentums and from inguinal fat pads, respectively being 0.72±0.05 days and 0.73±0.06 days (P> 0.05). The frequencies of ADMSCs exhibiting CD11b、CD29、CD106 and CD90 phenotype are similar (P> 0.05). The frequencies of ADMSCs exhibiting CD45 and CD49d is different (P<0.05).
3. The morphological characterization of these ADMSCs were almost the same in two groups. The yield of stromal vascular fraction cells and adherent cells from injured rats (respectively being 0.47±0.20×104/mg and 1.29±0.52×104/mg) were fewer than them from normal rats (respectively being.1.43±0.17×104/mg and 3.84±0.54×104/mg) (P<0.05). ADMSCs from injured and normal rats could differentiate into adipogenic and osteogenic lineages. The growth curves of these two kinds ADMSCs showed no obvious difference. The population doubling times based on the growth curves were similar, respectively being 1.11±0.06 days and 1.09±0.05 days (P> 0.05). The immunophenotypes analysis revealed that ADMSCs from two groups were the same positive for CD29 and CD90, but negative for CD11b, CD45, CD49d and CD106. The frequency of CD11b+、CD29+、CD45+、CD49d+、CD90+and CD106+ ADMSCs derived from injured rats and normal rats are similar (P>0.05).
4. Grafted ADMSCs improved cognitive function in rats after TBI.
Morris water maze tests:
All the rats were tested, not having obvious health problems such as weight lost. There was an overall significant effect of treatment on the escape latency throughout the behavioural experiment of the subjects (P<0.05). The escape latencies to platform in experimental group were significantly shorter than those in control group on days 1, 2,3 and 4. The escape latency was no significant difference among the three groups on the fifth day.
The migration of transplanted ADMSCs was determined by immunofluorescent staining of BrdU. In animals of experiment group we examined, many of the grafted ADMSCs were found in the injured cortex and were not found in the normal cortex and other districts.
The levels of BDNF and GDNF were significantly higher in experimental group than them in control group (P=0.000).
Conclusions
1. The ADMSCs harvested from SD rat obtained through an enzyme-based isolation procedures have the ability to differentiate along adipogenic and osteogenic lineages.. The results revealed the immunophenotypic characterization of them is consistent with mesenchymal stem cells.
2. The immunophenotypic characterizations of ADMSCs reserved in greater omentums and subcutaneous adipose tissues are not absolutely similar. They all have the ability to differentiate along adipogenic and osteogenic lineages. The quantitative osteogenesis assay and the quantitative adipogenesis assay are the same.
3. The yield of ADMSCs in injured rats is decreased and influenced by skin wound. ADMSCs cultured from injured and normal rats are similar in multiple differentiations, growth characteristics and cell surface markers.
4. Intravenous transplatation of ADMSCs results in functional recovery after TBI in rats. The grafted ADMSCs can migrate to the injured cortex. The cell transplantation results in a higher level of BDNF and GDNF in the injured brain.
引文
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