转SHH基因的骨髓间充质干细胞修复大鼠脊髓损伤的实验研究
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摘要
由于交通事故、外伤、塌方、自然灾害等原因导致的脊髓损伤（Spinal cordinjury, SCI）一直是医学界的难题之一。脊髓损伤可以导致神经细胞的缺失，破坏神经传导，进而引起许多组织、器官功能障碍，是最常见的一种致残、致死率性疾病。脊髓损伤不仅会给患者本人带来身体和心理的严重伤害，还会对整个社会造成巨大的经济负担，严重影响人类生活质量。
目前脊髓损伤的主要治疗方法包括手术解除压迫、类固醇、蛋白激酶、金属蛋白酶抑制和再生技术等，但是治疗效果有限。近年来，关于脊髓损伤后的修复治疗研究取得了很大进展，其中干细胞基因疗法移植治疗是公认的有希望的治疗方法，是目前治疗脊髓损伤的研究焦点。
研究表明骨髓间充质干细胞具有来源广泛、具有良好的增殖和分化能力、易于获得和培养、免疫排斥反应低，而且不涉及伦理问题，是理想的基因治疗的载体。在治疗脊髓损伤方面，骨髓间充质干细胞可以分泌神经营养因子，改善脊髓损伤的微环境；具有桥接作用，可以为轴突再生提供一个很好的连接通道；可以促进新生血管的形成并对损伤部位进行靶点归巢。然而由于脊髓损伤后局部缺血、低氧等微环境的改变，骨髓间充质干细胞移植后不能很好的存活，这极大程度上限制了它的应用。
生长因子对于脊髓损伤的修复必不可少。音猬因子（Sonic hedgehog，Shh）是胚胎发育过程中由脊索产生的一种因子，作为一种重要的调控因子Shh参与了调控了神经系统的发育和分化过程。当前研究表明Shh信号途径在胚胎干细胞分化为神经系统的过程中是必须的。它在以下方面对脊髓再生产生作用：音猬因子可以诱导干细胞生成运动神经元和少突胶质细胞,提高骨髓间充质干细胞分泌神经营养因子,促进神经元的存活和促进轴突的生长,阻碍星形胶质细胞的生成。然而因为Shh的快速清除性，Shh蛋白的注射并不能对脊髓损伤功能恢复产生很好的效果。
在我们的实验中，我们使用转染音猬因子基因的骨髓间充质干细胞来治疗脊髓损伤，它可以持续稳定的分泌和表达Shh，尝试着解决音猬因子快速清除性和骨髓间充质干细胞的生存问题，进而可以很好的促进脊髓损伤后功能的恢复。
本实验从细胞研究及动物实验两个方面进行了相关工作。研究内容主要包括以下三个部分：
1提取骨髓间充质干细胞并进行培养和鉴定。
2构建Shh载体，采用慢病毒技术转染骨髓间充质干细胞并进行鉴定。
3制作大鼠脊髓损伤模型，通过蛛网膜下腔注射转Shh的骨髓间充质干细胞，观察脊髓损伤后的修复效果。
第一部分大鼠骨髓间充质干细胞的分离、培养和鉴定研究目的
建立大鼠骨髓间充质干细胞的分离、培养，并进行细胞形态学、细胞增殖、细胞表面标记物、成骨成脂诱导分化的鉴定。研究方法
1.实验动物
健康雌性Sprague Dawley（SD）大鼠，体重160-200g,由北京军事医学科学院动物实验中心提供。
2．大鼠骨髓间充质干细胞（bone mesenchymal stem cells，BMSCs）的原代分离培养
10%水合氯醛腹腔麻醉SD大鼠，无菌条件下快速取大鼠的股骨及胫骨，PBS清洗干净。用剪刀剪掉长骨的骨骺端，露出骨髓腔，用含有100U/ml青、链霉素的L-DMEM培养基将骨髓冲出，直至骨髓腔发白；将冲出的骨髓内容物制成单细胞悬液，1200r/min离心5分钟，弃去上清，重悬细胞置于SD MSC完全培养基中生长，接种浓度为6-8×l06/ml，置于37°C、5%CO2的细胞培养箱中。
3.BMSCs的纯化及传代
在原代培养过程中，48小时后半量更换培养基，以后每3d全量更换含有10%胎牛血清的L-DMEM培养基，当细胞铺满培养皿约80%时，使用胰酶-EDTA溶液消化，1:3的比例进行传代培养。
4.BMSCs的形态学检查
培养后使用倒置显微镜观察贴壁细胞的形态变化以及生长情况，并进行拍照。
5.MTT法检测细胞增殖能力
取生长状态良好的SD大鼠BMSCs传至3代并接种于96孔培养板中，接种密度为5×103/孔，将细胞培养板置入浓度为5%的CO2培养箱中培养。每日取出一板采用MTT法进行检测。选择490nm波长，在酶联免疫检测仪上连续7日测定各孔光吸收值，并记录结果。以观测时间为横轴，光吸收值为纵轴绘制细胞生长曲线。
6.流式细胞术测定BMSCs的细胞表面标记物表达
取第3代生长状态良好细胞，胰酶-EDTA溶液消化，4°C离心，1200r/min，5min，计数细胞，各管依次加入单克隆抗体CD29, CD34, CD44, and CD45。室温孵育30min，PBS液洗涤细胞3次，除去未结合抗体，与FITC标记和PE标记的二抗避光作用30min， PBS液重悬细胞并置于冰上，流式细胞仪进行检测分析。
7.BMSCs的成骨成脂诱导分化能力
取第3代BMSCs，接种于6孔的细胞培养板中，待贴壁细胞密度达到80%时，在各诱导孔的完全培养液中分别加入成骨细胞诱导剂和成脂细胞诱导剂，各诱导孔每3天换液1次，同时设置未加诱导培养液的细胞培养孔作为空白对照。成骨细胞诱导剂诱导28天后，室温下40g/L多聚甲醛固定10分钟，然后对诱导分化的成骨细胞进行茜素红染色；成脂细胞诱导剂诱导23天后，40g/L多聚甲醛固定，然后对诱导分化的脂肪细胞进行油红O染色。倒置显微镜观察并拍照。
结果
1.BMSCs的形态学观测
SD大鼠骨髓间充质干细胞形态以梭形细胞为主，呈放射状细胞集落单位排列，细胞生长良好，可持续稳定传代10代以上。
2.BMSCs的细胞生长曲线
细胞生长曲线显示BMSCs符合正常细胞生长特征并且生长活跃。
3.BMSCs表面标记物的表达
第3代骨髓间充质干细胞CD29，CD44均呈阳性表达，而CD34，CD45呈阴性表达。
4.BMSCs成骨成脂诱导分化鉴定
成骨、成脂诱导分化后，茜素红染色和油红O染色均呈阳性。
小结
全骨髓贴壁培养法操作步骤简单，能够大量分离、纯化、扩增骨髓间充质干细胞，获得的细胞具有骨髓间充质干细胞的生物学特性，经诱导分化培养后具有成骨成脂分化潜能。实验所用的全骨髓贴壁法法为基因工程提供充足的载体细胞来源具有重要的现实意义。
第二部分构建转音猬因子基因的骨髓间充质干细胞和鉴定
研究目的
以慢病毒为载体，体外构建音猬因子基因转染的骨髓间充质干细胞，并对转染细胞的转染效率进行鉴定，为干细胞移植治疗脊髓损伤提供稳定可靠的细胞来源。
研究方法
1.构建大鼠音猬因子（Sonic hedgehog，Shh）的载体
首先利用重叠PCR扩增attB1-Shh-attB2；然后利用Gateway Technology构建pDown-Shh；最后使用Gateway Technology构建pLV.EX3d.P/puro-EF1α>Shh>IRES/DsRed（red fluorescent protein，红色荧光蛋白）Express2。
2.构建Shh慢病毒并利用其感染BMSCs
在脂质体Lipofectamine2000作用下转染293T细胞，逐孔滴度稀释法检测慢病毒滴度；以Shh慢病毒感染BMSCs，并通过荧光表达法判定Shh转染情况。
3.Real-time PCR测定转染细胞Shh的mRNA表达
细胞组织总RNA提取后，按照说明书将提取的RNA在一定的化学反应体系以及反转录条件下合成cDNA,在Real-time PCR仪上进行扩增，记录Ct值、扩增曲线以及溶解曲线。并相对定量计算实验结果（2-ΔΔCT）。
4.Western blot测定转染细胞Shh的蛋白含量
细胞组织提取蛋白质后，用BCA法测定蛋白浓度。配置12%凝胶，上样、电泳、转膜、封闭、一抗反应、二抗反应，化学发光检测。
结果
1.构建转音猬因子基因的骨髓间充质干细胞
构建音猬因子的重组慢病毒载体，经酶切及测序方法鉴定完全正确，可以转染293T细胞并表达，滴度为2-4×108TU/ml，转染BMSCs5日后红色荧光蛋白表达显著增强。
2.转Shh基因的骨髓间充质干细胞Shh表达增高
同未转染Shh的骨髓间充质干细胞相比，Shh慢病毒感染BMSCs能持续稳定高水平表达Shh mRNA和蛋白。
小结
成功构建Shh的慢病毒，然后将Shh基因成功转染至BMSCs基因组中，导致Shh的持续稳定高水平表达。为接下来的体内实验提供了基础。
第三部分音猬因子转染的骨髓间充质干细胞修复脊髓损伤的效果
研究目的
建立统一的大鼠脊髓损伤模型，然后在大鼠脊髓损伤后的7天将转音猬因子（Sonic hedgehog，Shh）的骨髓间充质干细胞移植入大鼠脊髓损伤模型，观察骨髓间充质干细胞的存活情况和脊髓损伤的运动功能的恢复。
研究方法
1.实验动物
健康雌性Sprague Dawley（SD）大鼠，体重200-220g,由北京军事医学科学院动物实验中心提供。
2.建立大鼠脊髓损伤实验动物模型
实验动物10%水合氯醛麻醉（0.3ml/100g,腹腔注射）；然后采用改良的ALLEN打击法。以T10为标志，行椎板切除术，暴露脊髓硬膜囊，垫一塑料垫片，将10g中的打击杆自25mm高度下落25mm(打击杆底部直径2.5mm)撞击脊髓。
3.蛛网膜下腔注射转音猬因子的大鼠骨髓间充质干细胞（Shh-BMSCs）治疗脊髓损伤
在脊髓损伤造模成功后7天，在大鼠L4节段咬除椎板，通过蛛网膜下腔将Shh-BMSCs移植入大鼠脊髓损伤模型，注射BMSCs和PBS作为对照。
4.Shh-BMSCs治疗脊髓损伤后Shh的蛋白测定
大鼠脊髓损伤治疗后28日，通过Western blot法检测脊髓组织Shh蛋白水平的变化。
5.Shh-BMSCs对脊髓损伤大鼠神经保护作用的测定
5.1神经营养因子的表达
大鼠脊髓损伤治疗后28日，通过Western blot法检测脊髓组织成纤维细胞生长因子和血管内皮生长因子蛋白水平的变化。
5.2BMSCs的存活情况
大鼠脊髓损伤治疗后28日，损伤中心冰冻切片使用荧光显微镜观察，ImageJ软件进行BMSCs数量的分析
5.3神经丝蛋白200（NF200）和胶质纤维酸性蛋白（GFAP）免疫荧光染色的测定
大鼠脊髓损伤治疗后28日，损伤中心周围2mm的冰冻切片进行免疫荧光染色鉴定，经过洗片、破膜、封闭、一抗孵育（神经丝蛋白200和胶质纤维酸性蛋白）、二抗孵育和DAPI染色，最后使用荧光显微镜观察。使用Image J软件进行荧光表达量的分析。
5.4尼氏染色对脊髓前角运动神经元进行检测
大鼠脊髓损伤治疗后28日，损伤中心冰冻切片进行尼氏染色，经过预热60°C1%的甲苯胺蓝染液染色40min、蒸馏水洗3次、95%酒精快速分化脱色、无水乙醇快速脱水，二甲苯透明，通风橱中风干后中性树胶封片。倒置显微镜下照相并对脊髓前角运动神经元进行计数，使用Image J软件进行人工定量。
5.5HE染色对损伤空洞进行评估
大鼠脊髓损伤后28日，损伤中心冰冻切片进行HE染色，Harris苏木素染液染色7分钟，0.5％伊红(水溶性)染液染色2-4分钟，倒置显微镜下照相并对损伤空洞体积进行评估，使用Image J软件进行定量分析。
6.Shh-BMSCs对脊髓损伤大鼠行为学影响的评定
脊髓损伤术后每一周对大鼠的运动情况进行Basso–Beattie–Bresnahan(BBB)评分，总共6周。由3位实验人员了解评分标准后，采用单盲法进行评分，取测量评分的平均值。
7.统计处理
实验数据以均数±标准差（Mean±SD）表示，多组间均数比较采用单因素方差分析，BBB评分采用重复测量的方差分析。采用SPSS15.0统计软件对数据进行统计分析。P<0.05被认为具有统计学意义。
结果
1.脊髓损伤大鼠模型建立成功
大鼠脊髓损伤后迅速出现摇尾反射，双后肢及躯体回缩，而后呈迟缓性瘫痪，表明脊髓损伤造模成功。术后腹腔注射青霉素并进行膀胱按摩。所有大鼠术后均存活。
2.Shh-BMSCs促进体内Shh蛋白的表达
大鼠脊髓损伤治疗28日，通过Western blot法检测脊髓组织中Shh的蛋白表达。经过Shh-BMSCs的治疗后，Shh的蛋白水平表达增高，差异有统计学意义。
3.Shh-BMSCs能够促进神经保护
3.1Shh-BMSCs促进成纤维细胞生长因子和血管内皮生长因子蛋白的表达
大鼠脊髓损伤治疗28日，通过Western blot法检测脊髓组织中成纤维细胞生长因子和血管内皮生长因子的蛋白表达。经过Shh-BMSCs的治疗后，成纤维细胞生长因子和血管内皮生长因子的蛋白水平明显高于BMSCs和PBS治疗，BMSC组蛋白表达高于PBS组，差异有统计学意义。
3.2Shh-BMSCs促进BMSCs的存活
大鼠脊髓损伤治疗28日，在荧光显微镜下观察BMSCs的存活情况。Shh-BMSCs组BMSCs的数量明显高于BMSCs组，差异有统计学意义。
3.3Shh-BMSCs促进NF200的表达，抑制GFAP的表达
大鼠脊髓损伤治疗28日，通过免疫荧光法检测脊髓组织中NF200和GFAP的荧光表达。Shh-BMSCs组NF200的荧光表达水平明显高于BMSCs组和PBS组，BMSCs组NF200的荧光表达水平高于PBS组,差异有统计学意义，Shh-BMSCs组GFAP的荧光表达水平明显低于BMSCs组和PBS组，差异有统计学意义，BMSCs组和PBS组的GFAP的荧光表达差异没有有统计学意义。
3.4Shh-BMSCs增加了大鼠脊髓损伤后脊髓前角运动神经元的数量
大鼠脊髓损伤治疗28日，通过尼氏染色检测脊髓组织中脊髓前角运动神经元的数量。同BMSCs组和PBS组相比，Shh-BMSCs组脊髓前角运动神经元数量明显增多，BMSCs组脊髓前角运动神经元数量多于PBS组，差异有统计学意义。
3.5Shh-BMSCs减少了大鼠脊髓损伤后空洞的面积
大鼠脊髓损伤治疗28日，通过HE染色检测脊髓损伤后空洞面积的变化。同BMSCs组和PBS组相比，Shh-BMSCs组脊髓损伤空洞面积明显缩小，BMSCs组脊髓空洞面积小于PBS组，差异有统计学意义。
4.Shh-BMSCs促进运动功能的康复
在BBB评分期间，从2周到6周，Shh-BMSCs组大鼠的BBB评分明显高于BMSCs组和PBS组，BMSCs组明显高于PBS组，差异有统计学意义。
小结
Shh-BMSCs在治疗脊髓损伤的过程中，Shh可以持续分泌，Shh表达增强，促进了BMSCs的存活，增强了神经的保护和运动功能的康复，能够促进脊髓损伤后神经功能的恢复，可以为临床治疗脊髓损伤提供更好的方法。
Spinal cord injury (SCI) is one of the problems of the medical challenge, whichdue to injury, traffic accident, landslides, natural disasters and etc. SCI, which canlead to loss of cells and damage of neural tracts, therefore result in many tissular andorganic dysfunction, is one of the most common disability and lethal disease. Patientsafter SCI will not only bring serious physical and mental damage, but also cause ahuge economic burden to the whole society, therefore seriously affect the quality ofhuman life.
At present the main treatment of spinal cord injury include surgery removingoppression, steroids, protein kinase, metalloproteinases, suppression and regenerationtechnology and etc, however the effect of treatment is limited. In recent years, how torepair spinal cord injury has achieved great progress. Stem cell gene transplantation isrecognized as a promising treatment, and is the focus for the treatment of spinal cordinjury.
Previous studies have shown that bone marrow mesenchymal stem cells(BMSCs), which have widely source, a good ability of proliferation anddifferentiation, easy to obtain and cultivation, low immune rejection, and don’tinvolve the ethical issues, are the ideal carrier of the gene therapy. In their treatmentfor spinal cord injury, BMSCs can secrete neurotrophic factors, improve themicroenvironment of the spinal cord injury, differentiate into neurons which replacenecrosis or death of nerve cells, have the bridge role which can provide the axonregeneration with a good connection channel, promote the formation of new blood vessels and target spinal cord lesions. However, most BMSCs die aftertransplantation as a result of ischemia and hypoxia in the region of SCI.
Growth factor is essential for repairing spinal cord injury. Sonic hedgehog (Shh),which is produced by the notochord and floor plate, is a member of the family ofhedgehog proteins and is essential for the development and differentiation of thenervous system. Shh works on several aspects of spinal cord regeneration: it inducesthe production of motor neurons and oligodendrocytes, enhances the delivery ofneurotrophic factor to improve the microenvironment in BMSCs, facilitates neuronalsurvival, promotes axonal growth, and prevents activation of the astrocyte lineageafter SCI. However, an injection of Shh protein after SCI does not have a good effecton functional behavioral recovery because of the fast clearance of Shh protein fromthe spinal cord.
In our study, we speculated that transplantation of BMSCs transfected with Shh(Shh-BMSCs), which can provide long-term stable expression and secretion of highlevels of Shh, could conquer the difficulties of inherent in treatment with Shh alone,to facilitate repair and recovery after SCI.
In this study we referred to cell and animal research. The research mainly coversthe following three parts:
1. To isolate, culture and identification of rat BMSCs
2. To construct Shh carrier, use the lentiviral vector technology to transfect BMSCsand identify the transduction effect.
3To establish rat spinal cord injury model, treat SCI using Shh-BMSCs via thesubarachnoid injection and observe the therapeutic effects after SCI.
Section one Isolation, culture and identification of rat bonemarrow mesenchymal stem cells
Objective
To establish a method of isolation, cultivation of rat bone marrow mesenchymalstem cells (BMSCs) in vitro, to identify cell morphology, cell proliferation, cellsurface markers, osteogenetic and adipogenic differentiation capacity.
Research methods
1.Animals
Adult female Sprague Dawley (SD) rats,160-200g, were purchased fromAnimal Center of Chinese People’s Army Military Medical and Scientific Academy,Beijing, China.
2. Isolation of BMSCs and cultivation of BMSCs in primary
culture
Animals were anesthetized with10%chloral hydrate. Take the rat femur andtibia rapidly and wash them using PBS. Use scissor to cut off the epiphyseal end,expose the long bone marrow cavity, and wash them using L-DMEM mediumcontaining100U/ml penicillin streptomycin until the marrow cavity was white. Thesingle cell suspension was centrifuged by1200r/min for5minutes and cultured bySD MSC complete medium at37°C under a5%CO2atmosphere. The inoculationconcentration is6-8×l06/ml.
3. The cultivation, purification and passage of BMSCs
In the process of primitive culture, change a half quantity medium after48hoursand follow a full volume replacement every three days. When BMSCs were coveredabout80%, use trypsin-EDTA solution to digest them. The ratio of subculture was1:3.
4. The cell morphology of BMSCs
To observe the cell morphology and growth condition using inverted microscopeand photograph them.
5. To detect cell proliferation using MTT methods
At passage3, BMSCs were cultured in96-well culture plate under a5%CO2atmosphere. The inoculation density was5x103/hole. Take out one plate and detectit every day by MTT method. Detect the optical density of palte for seven days usingenzyme-linked immune detector absorption value, and the wavelength was490nm.The observation time was for the horizontal axis and the value of optical density wasthe vertical axis.
6. To detect the cell surface markers of BMSCs using flowcytometric analysis
At passage3, BMSCs were trypsinized and centrifuged by1200r/min for5minutes at4°C. Count the cells and join the monoclonal antibody CD29, CD34,CD44, and CD45for30min at room temperature. After washing cells using PBS toremove the uncombined antibody, join the second antibody labeling FITC and PE in adark place for30min, and detect them using flow cytometric analysis.
7. Osteogenetic and adipogenic differentiation capacity ofBMSCs
At passage3, BMSCs were cultured in6-well culture plate. When BMSCs werecovered about80%, join the osteogenic and adipogenic media and change a fullvolume replacement every three days, at the same time set up a blank control withoutjoining induction media. After cells was fixed by40g/L paraformaldehyde at roomtemperature for10minutes, we evaluated osteogenic differentiation by alizarin redstaining at day28and the adipogenic differentiation was assessed at day23bystaining with oil red-O solution. Observe and photograph them using inverted microscope.
Results
1. The morphological observation of BMSCs
BMSCs were spindle and showed radial colony arrangement. Cells kept goodgrowth and could passage in continuous over10passages.
2. The cell growth curve of BMSCs
Cell growth curve demonstrated that BMSCs were consistent with the growthcharacteristics and strong activity of normal cells.
3. The expression of BMSCs cell surface markers
At passage3, BMSCs were found to express cell markers CD29and CD44, butCD34and CD45were not detected.
4. The identification of osteogenic and adipogenicdifferentiation
After osteogenic and adipogenic differentiation, alizarin red and oil red-Ostaining were positive.
Summary
The whole bone marrow adherence method is simple, and can isolate, purify andamplify BMSCs in vitro. The obtained cells have general biologicalcharacteristics ofbone mesenchymal stem cells, and also have potentiality of osteogenic andadipogenic differentiation. This experimental way has important practicalsignificance to provide suffient source of carrier cells for gene engineering.
Section two Construction of bone marrow-derivedmesenchymal stem cells expressing the Shh transgene andidentification
Objective
To construct the Shh gene modified BMSCs using a lentiviral vector in vitro andthen observe the identification of transduction efficiency, provide a potential stablestem cell source for the treatment of SCI.
Research methods
1. Construction of Shh carrier
Firstly amplify attB1-Shh-attB2using overlapping PCR; then constructpDown-Shh using Gateway Technology; finally structure pLV.EX3d.P/puro-EF1α>Shh>IRES/DsRed(red fluorescent protein)Express2using Gateway Technology.
2. Constuction of lentiviral vector and BMSCs expressing theShh transgene using them
Transfection of293T cells using Lipofectamine2000. Detect the titers oflentivirus using hole-by-hole dilution method. Infect BMSCs with shh lentiviralvector and detect the transfection efficiency through the fluorescence.
3. Detect the shh mRNAexpression of Shh-BMSCs usingReal-time PCR analysis
Total RNA was extracted from the cells. Total RNA was reverse transcribed intocDNA under a PCR-reaction system and the condition of Reverse transcription.Record the value of Ct, amplification curve and dissolve curve. Calculate theexperimental results (2-ΔΔCT) using relative quantitative methods.
4. Detect the shh proteinn expression of Shh-BMSCs usingWestern blot analysis
After the proteins were collected from cells, protein concentrations were testedby the BCA method. Configuration of12%SDS-polyacrylamide gel, on sample,electrophoresis, transfer the membrane, blocked, reaction of primary and secondaryantibody, electrochemiluminescence detection.
Results
1. Construction of BMSCs expressing the Shh transgene
The shh lentiviral vector was constructed. It was entirely correct by enzymedigestion and sequencing identification, and it could transfect293T cells and achieveexpression. The lentiviral titer was2-4×108TU/ml. The fluorescence expressionincreased significantly at5d following transduction
2. Shh-BMSCs increased the shh expression
Shh-BMSCs could long-term stable mRNA and protein expression of high levelsof Shh using lentiviral vector comparing with the uninfected BMSCs.
Summary
Construction of Shh lentiviral vector successfully and Shh-BMSCs, which canlong-term stable expression of high levels of Shh. BMSCs were the basis for thesubsequent experiments.
Section three Effect of bone marrow-derived mesenchymalstem cells expressing the Shh transgene on recovery after spinalcord injury in rats
Objective
To establish the spinal cord injury model and observe the BMSCs survival andfunctional recovery via transplantation of Shh-BMSCs at7days after SCI.
Research methods
1.Animals
Adult female Sprague Dawley (SD) rats,200-220g, were purchased fromAnimal Center of Chinese People’s Army Military Medical and Scientific Academy,Beijing, China.
2. Spinal cord injury model
Animals were anesthetized with10%chloral hydrate (0.3ml/100g,intraperitoneal injection); then followed by Allen’s methods. In brief, the spinal cordwas exposed by performing T10laminectomy. SCI was induced by dropping a10-grod (diameter,2.5mm) from a height of25mm onto an impounder positioned on thespinal cord.
3. Treatment of Shh-BMSCs after SCI via intrathecal injection
Seven days after SCI, a second laminectomy was performed at lumbar level4.We therefore microinjected Shh-BMSCs into the model rats intrathecally. In thecontrol group, BMSCs or PBS was injected.
4. Detect the shh protein expression after treatment ofShh-BMSCs in rats with SCI
To detect the shh protein expression using Western blot analysis at28d aftertreatment.
5. Detect the neuroprotection after treatment of Shh-BMSCs inrats with SCI
5.1The expression of neurotrophic factors
To detect the basic fibroblast growth factor (bFGF) and vascular endothelialgrowth factor (VEGF) protein expression using Western blot analysis at28d aftertreatment.
5.2Shh-BMSCs promoted the BMSCs survival
To observe the lesion epicenter using a fluorescence microscope at28d aftertreatment and analyze the number of BMSCs using Image J software.
5.3Detection of neurofilament200(NF200) and glial fibrillaryacidic protein (GFAP) immunofluorescence staining
The sections around the lesion epicenter were washed, blocked, incubated byprimary antibodies (NF200and GFAP) and secondary antibody and stained by DAPIat28d after treatment. The expressions of GFAP and NF200were observed using afluorescence microscope and analyzed by Image J software.
5.4Nissl’s staining for the motoneurons
The sections around the lesion epicenter were stained in1%solvent blue at60°Cfor40min, washed three times using distilled water, differentiated in95%ethanol,dehydrated in100%ethanol,cleared in xylene and covered by using a resinousmedium. Photograph and count the motoneurons using a inverted microscope.Analyze them by Image J software.
5.5Evaluate the lesion cavity using HE staining
The sections around the lesion epicenter were stained in Harris hematoxylin for7min and0.5%eosin for2-4min. The volumes of lesion cavity were photographedusing a fluorescence microscope and analyzed by Image J software.
6. Evaluate the behavioral recovery after treatment ofShh-BMSCs in rats with SCI
The Basso–Beattie–Bresnahan (BBB) locomotor rating score is widely used toevaluate hind-limb motor function. Rats were examined by three trained examinersfor6weeks after SCI in a double-blinded manner. The BBB results were averaged.
7. Statistical analysis
All data were expressed as mean±SD. One-way analysis of variance (ANOVA)was used to compare mean values. A repeated measure ANOVA was used to analyzeweekly BBB scores in different groups. Statistical analyses were performed usingSPSS15.0software. Statistical evaluations were considered significant at P <0.05.
Results
1. The model of spinal cord injury was successfully established
Rat tail swing and hind limb extremity paralysis indicated that induction of SCIwas successful. Manual bladder expression was performed before recovery ofautonomous urination. All rats were injected penicillin intraperitoneally and survivedbetter.
2. Shh-BMSCs promoted the expression of Shh in vivo
Shh expressions of spinal cord were detected by Western blot analysis at28dafter treatment. The protein expression of Shh was significantly greater afterShh-BMSCs treatment than the other two interruptions. Differences were consideredstatistically significant (P <0.05).
3. Shh-BMSCs increased the neuroprotection
3.1Shh-BMSCs promoted the expression of bFGF and VEGF
The bFGF and VEGF protein levels were determined using western blot analysisat day28after treatment.The protein levels of bFGF and VEGF in the Shh-BMSCsgroup were markedly higher than those in the PBS and BMSCs groups, and the bFGFand VEGF protein expression in the BMSCs group increased significantly by day28in comparison with the levels in the PBS group. Differences were consideredstatistically significant (P <0.05).
3.2Shh-BMSCs enhanced the BMSCs survival
BMSCs survival was detected using a fluorescence microscope at28d aftertreatment. The number of BMSCs present at this time point in the Shh-BMSCs groupwas significantly more than those in the BMSCs group on day28after transplantation.Differences were considered statistically significant (P <0.05).
3.3Shh-BMSCs increased the expression of NF200and reducedthe expression of GFAP
The expressions of NF200and GFAP were detected using immunofluorescencestaining. On day28after transplantation, the expression of NF200in the Shh-BMSCsgroup was significantly higher than that in the other two groups, and the staining inthe PBS group was significantly lower than that in the BMSCs group. The expressionof GFAP on day28after transplantation was markedly weaker in the Shh-BMSCsgroup versus the other two groups, and the staining between the PBS and BMSCsgroups did not differ significantly. Differences were considered statisticallysignificant (P <0.05).
3.4Shh-BMSCs increased the number of motoneurons after SCI
Nissl’s staining at the ventral horns was used to evaluate the number ofmotoneurons at28d after treatment. Injection of Shh-BMSCs significantly increasedthe number of motoneurons in the ventral horns, compared with injection of BMSCsor PBS. The BMSCs group had more motoneurons than the PBS group at the ventralhorns. Differences were considered statistically significant (P <0.05).
3.5Shh-BMSCs reduced the area of lesion cavity after SCI
HE staining was used to evaluate the area of lesion cavityat28d after treatment.Injection of Shh-BMSCs significantly reduced the area of lesion cavity, comparedwith injection of BMSCs or PBS. The BMSCs group had less area of lesion cavitythan the PBS group. Differences were considered statistically significant (P <0.05).
4. Shh-BMSCs promoted significant functional recovery
BBB tests showed highly significant motor improvement in rats that had beentreated with Shh-BMSCs compared with those treated with PBS or BMSCs, from2wto6w after SCI. The BBB score of the BMSCs group was significantly greater thanthose of the PBS group (P <0.05).Differences were considered statisticallysignificant.
Summary
BMSCs that have been transfected with a Shh transgene, which can providelong-term stable expression and secretion of high levels of Shh and so enhance thesurvival of BMSCs, would promote the neuroprotection and facilitate repair andrecovery after SCI and could be a potential valuable therapeutic intervention for SCIclinically.
目前脊髓损伤的主要治疗方法包括手术解除压迫、类固醇、蛋白激酶、金属蛋白酶抑制和再生技术等，但是治疗效果有限。近年来，关于脊髓损伤后的修复治疗研究取得了很大进展，其中干细胞基因疗法移植治疗是公认的有希望的治疗方法，是目前治疗脊髓损伤的研究焦点。
研究表明骨髓间充质干细胞具有来源广泛、具有良好的增殖和分化能力、易于获得和培养、免疫排斥反应低，而且不涉及伦理问题，是理想的基因治疗的载体。在治疗脊髓损伤方面，骨髓间充质干细胞可以分泌神经营养因子，改善脊髓损伤的微环境；具有桥接作用，可以为轴突再生提供一个很好的连接通道；可以促进新生血管的形成并对损伤部位进行靶点归巢。然而由于脊髓损伤后局部缺血、低氧等微环境的改变，骨髓间充质干细胞移植后不能很好的存活，这极大程度上限制了它的应用。
生长因子对于脊髓损伤的修复必不可少。音猬因子（Sonic hedgehog，Shh）是胚胎发育过程中由脊索产生的一种因子，作为一种重要的调控因子Shh参与了调控了神经系统的发育和分化过程。当前研究表明Shh信号途径在胚胎干细胞分化为神经系统的过程中是必须的。它在以下方面对脊髓再生产生作用：音猬因子可以诱导干细胞生成运动神经元和少突胶质细胞,提高骨髓间充质干细胞分泌神经营养因子,促进神经元的存活和促进轴突的生长,阻碍星形胶质细胞的生成。然而因为Shh的快速清除性，Shh蛋白的注射并不能对脊髓损伤功能恢复产生很好的效果。
在我们的实验中，我们使用转染音猬因子基因的骨髓间充质干细胞来治疗脊髓损伤，它可以持续稳定的分泌和表达Shh，尝试着解决音猬因子快速清除性和骨髓间充质干细胞的生存问题，进而可以很好的促进脊髓损伤后功能的恢复。
本实验从细胞研究及动物实验两个方面进行了相关工作。研究内容主要包括以下三个部分：
1提取骨髓间充质干细胞并进行培养和鉴定。
2构建Shh载体，采用慢病毒技术转染骨髓间充质干细胞并进行鉴定。
3制作大鼠脊髓损伤模型，通过蛛网膜下腔注射转Shh的骨髓间充质干细胞，观察脊髓损伤后的修复效果。
第一部分大鼠骨髓间充质干细胞的分离、培养和鉴定研究目的
建立大鼠骨髓间充质干细胞的分离、培养，并进行细胞形态学、细胞增殖、细胞表面标记物、成骨成脂诱导分化的鉴定。研究方法
1.实验动物
健康雌性Sprague Dawley（SD）大鼠，体重160-200g,由北京军事医学科学院动物实验中心提供。
2．大鼠骨髓间充质干细胞（bone mesenchymal stem cells，BMSCs）的原代分离培养
10%水合氯醛腹腔麻醉SD大鼠，无菌条件下快速取大鼠的股骨及胫骨，PBS清洗干净。用剪刀剪掉长骨的骨骺端，露出骨髓腔，用含有100U/ml青、链霉素的L-DMEM培养基将骨髓冲出，直至骨髓腔发白；将冲出的骨髓内容物制成单细胞悬液，1200r/min离心5分钟，弃去上清，重悬细胞置于SD MSC完全培养基中生长，接种浓度为6-8×l06/ml，置于37°C、5%CO2的细胞培养箱中。
3.BMSCs的纯化及传代
在原代培养过程中，48小时后半量更换培养基，以后每3d全量更换含有10%胎牛血清的L-DMEM培养基，当细胞铺满培养皿约80%时，使用胰酶-EDTA溶液消化，1:3的比例进行传代培养。
4.BMSCs的形态学检查
培养后使用倒置显微镜观察贴壁细胞的形态变化以及生长情况，并进行拍照。
5.MTT法检测细胞增殖能力
取生长状态良好的SD大鼠BMSCs传至3代并接种于96孔培养板中，接种密度为5×103/孔，将细胞培养板置入浓度为5%的CO2培养箱中培养。每日取出一板采用MTT法进行检测。选择490nm波长，在酶联免疫检测仪上连续7日测定各孔光吸收值，并记录结果。以观测时间为横轴，光吸收值为纵轴绘制细胞生长曲线。
6.流式细胞术测定BMSCs的细胞表面标记物表达
取第3代生长状态良好细胞，胰酶-EDTA溶液消化，4°C离心，1200r/min，5min，计数细胞，各管依次加入单克隆抗体CD29, CD34, CD44, and CD45。室温孵育30min，PBS液洗涤细胞3次，除去未结合抗体，与FITC标记和PE标记的二抗避光作用30min， PBS液重悬细胞并置于冰上，流式细胞仪进行检测分析。
7.BMSCs的成骨成脂诱导分化能力
取第3代BMSCs，接种于6孔的细胞培养板中，待贴壁细胞密度达到80%时，在各诱导孔的完全培养液中分别加入成骨细胞诱导剂和成脂细胞诱导剂，各诱导孔每3天换液1次，同时设置未加诱导培养液的细胞培养孔作为空白对照。成骨细胞诱导剂诱导28天后，室温下40g/L多聚甲醛固定10分钟，然后对诱导分化的成骨细胞进行茜素红染色；成脂细胞诱导剂诱导23天后，40g/L多聚甲醛固定，然后对诱导分化的脂肪细胞进行油红O染色。倒置显微镜观察并拍照。
结果
1.BMSCs的形态学观测
SD大鼠骨髓间充质干细胞形态以梭形细胞为主，呈放射状细胞集落单位排列，细胞生长良好，可持续稳定传代10代以上。
2.BMSCs的细胞生长曲线
细胞生长曲线显示BMSCs符合正常细胞生长特征并且生长活跃。
3.BMSCs表面标记物的表达
第3代骨髓间充质干细胞CD29，CD44均呈阳性表达，而CD34，CD45呈阴性表达。
4.BMSCs成骨成脂诱导分化鉴定
成骨、成脂诱导分化后，茜素红染色和油红O染色均呈阳性。
小结
全骨髓贴壁培养法操作步骤简单，能够大量分离、纯化、扩增骨髓间充质干细胞，获得的细胞具有骨髓间充质干细胞的生物学特性，经诱导分化培养后具有成骨成脂分化潜能。实验所用的全骨髓贴壁法法为基因工程提供充足的载体细胞来源具有重要的现实意义。
第二部分构建转音猬因子基因的骨髓间充质干细胞和鉴定
研究目的
以慢病毒为载体，体外构建音猬因子基因转染的骨髓间充质干细胞，并对转染细胞的转染效率进行鉴定，为干细胞移植治疗脊髓损伤提供稳定可靠的细胞来源。
研究方法
1.构建大鼠音猬因子（Sonic hedgehog，Shh）的载体
首先利用重叠PCR扩增attB1-Shh-attB2；然后利用Gateway Technology构建pDown-Shh；最后使用Gateway Technology构建pLV.EX3d.P/puro-EF1α>Shh>IRES/DsRed（red fluorescent protein，红色荧光蛋白）Express2。
2.构建Shh慢病毒并利用其感染BMSCs
在脂质体Lipofectamine2000作用下转染293T细胞，逐孔滴度稀释法检测慢病毒滴度；以Shh慢病毒感染BMSCs，并通过荧光表达法判定Shh转染情况。
3.Real-time PCR测定转染细胞Shh的mRNA表达
细胞组织总RNA提取后，按照说明书将提取的RNA在一定的化学反应体系以及反转录条件下合成cDNA,在Real-time PCR仪上进行扩增，记录Ct值、扩增曲线以及溶解曲线。并相对定量计算实验结果（2-ΔΔCT）。
4.Western blot测定转染细胞Shh的蛋白含量
细胞组织提取蛋白质后，用BCA法测定蛋白浓度。配置12%凝胶，上样、电泳、转膜、封闭、一抗反应、二抗反应，化学发光检测。
结果
1.构建转音猬因子基因的骨髓间充质干细胞
构建音猬因子的重组慢病毒载体，经酶切及测序方法鉴定完全正确，可以转染293T细胞并表达，滴度为2-4×108TU/ml，转染BMSCs5日后红色荧光蛋白表达显著增强。
2.转Shh基因的骨髓间充质干细胞Shh表达增高
同未转染Shh的骨髓间充质干细胞相比，Shh慢病毒感染BMSCs能持续稳定高水平表达Shh mRNA和蛋白。
小结
成功构建Shh的慢病毒，然后将Shh基因成功转染至BMSCs基因组中，导致Shh的持续稳定高水平表达。为接下来的体内实验提供了基础。
第三部分音猬因子转染的骨髓间充质干细胞修复脊髓损伤的效果
研究目的
建立统一的大鼠脊髓损伤模型，然后在大鼠脊髓损伤后的7天将转音猬因子（Sonic hedgehog，Shh）的骨髓间充质干细胞移植入大鼠脊髓损伤模型，观察骨髓间充质干细胞的存活情况和脊髓损伤的运动功能的恢复。
研究方法
1.实验动物
健康雌性Sprague Dawley（SD）大鼠，体重200-220g,由北京军事医学科学院动物实验中心提供。
2.建立大鼠脊髓损伤实验动物模型
实验动物10%水合氯醛麻醉（0.3ml/100g,腹腔注射）；然后采用改良的ALLEN打击法。以T10为标志，行椎板切除术，暴露脊髓硬膜囊，垫一塑料垫片，将10g中的打击杆自25mm高度下落25mm(打击杆底部直径2.5mm)撞击脊髓。
3.蛛网膜下腔注射转音猬因子的大鼠骨髓间充质干细胞（Shh-BMSCs）治疗脊髓损伤
在脊髓损伤造模成功后7天，在大鼠L4节段咬除椎板，通过蛛网膜下腔将Shh-BMSCs移植入大鼠脊髓损伤模型，注射BMSCs和PBS作为对照。
4.Shh-BMSCs治疗脊髓损伤后Shh的蛋白测定
大鼠脊髓损伤治疗后28日，通过Western blot法检测脊髓组织Shh蛋白水平的变化。
5.Shh-BMSCs对脊髓损伤大鼠神经保护作用的测定
5.1神经营养因子的表达
大鼠脊髓损伤治疗后28日，通过Western blot法检测脊髓组织成纤维细胞生长因子和血管内皮生长因子蛋白水平的变化。
5.2BMSCs的存活情况
大鼠脊髓损伤治疗后28日，损伤中心冰冻切片使用荧光显微镜观察，ImageJ软件进行BMSCs数量的分析
5.3神经丝蛋白200（NF200）和胶质纤维酸性蛋白（GFAP）免疫荧光染色的测定
大鼠脊髓损伤治疗后28日，损伤中心周围2mm的冰冻切片进行免疫荧光染色鉴定，经过洗片、破膜、封闭、一抗孵育（神经丝蛋白200和胶质纤维酸性蛋白）、二抗孵育和DAPI染色，最后使用荧光显微镜观察。使用Image J软件进行荧光表达量的分析。
5.4尼氏染色对脊髓前角运动神经元进行检测
大鼠脊髓损伤治疗后28日，损伤中心冰冻切片进行尼氏染色，经过预热60°C1%的甲苯胺蓝染液染色40min、蒸馏水洗3次、95%酒精快速分化脱色、无水乙醇快速脱水，二甲苯透明，通风橱中风干后中性树胶封片。倒置显微镜下照相并对脊髓前角运动神经元进行计数，使用Image J软件进行人工定量。
5.5HE染色对损伤空洞进行评估
大鼠脊髓损伤后28日，损伤中心冰冻切片进行HE染色，Harris苏木素染液染色7分钟，0.5％伊红(水溶性)染液染色2-4分钟，倒置显微镜下照相并对损伤空洞体积进行评估，使用Image J软件进行定量分析。
6.Shh-BMSCs对脊髓损伤大鼠行为学影响的评定
脊髓损伤术后每一周对大鼠的运动情况进行Basso–Beattie–Bresnahan(BBB)评分，总共6周。由3位实验人员了解评分标准后，采用单盲法进行评分，取测量评分的平均值。
7.统计处理
实验数据以均数±标准差（Mean±SD）表示，多组间均数比较采用单因素方差分析，BBB评分采用重复测量的方差分析。采用SPSS15.0统计软件对数据进行统计分析。P<0.05被认为具有统计学意义。
结果
1.脊髓损伤大鼠模型建立成功
大鼠脊髓损伤后迅速出现摇尾反射，双后肢及躯体回缩，而后呈迟缓性瘫痪，表明脊髓损伤造模成功。术后腹腔注射青霉素并进行膀胱按摩。所有大鼠术后均存活。
2.Shh-BMSCs促进体内Shh蛋白的表达
大鼠脊髓损伤治疗28日，通过Western blot法检测脊髓组织中Shh的蛋白表达。经过Shh-BMSCs的治疗后，Shh的蛋白水平表达增高，差异有统计学意义。
3.Shh-BMSCs能够促进神经保护
3.1Shh-BMSCs促进成纤维细胞生长因子和血管内皮生长因子蛋白的表达
大鼠脊髓损伤治疗28日，通过Western blot法检测脊髓组织中成纤维细胞生长因子和血管内皮生长因子的蛋白表达。经过Shh-BMSCs的治疗后，成纤维细胞生长因子和血管内皮生长因子的蛋白水平明显高于BMSCs和PBS治疗，BMSC组蛋白表达高于PBS组，差异有统计学意义。
3.2Shh-BMSCs促进BMSCs的存活
大鼠脊髓损伤治疗28日，在荧光显微镜下观察BMSCs的存活情况。Shh-BMSCs组BMSCs的数量明显高于BMSCs组，差异有统计学意义。
3.3Shh-BMSCs促进NF200的表达，抑制GFAP的表达
大鼠脊髓损伤治疗28日，通过免疫荧光法检测脊髓组织中NF200和GFAP的荧光表达。Shh-BMSCs组NF200的荧光表达水平明显高于BMSCs组和PBS组，BMSCs组NF200的荧光表达水平高于PBS组,差异有统计学意义，Shh-BMSCs组GFAP的荧光表达水平明显低于BMSCs组和PBS组，差异有统计学意义，BMSCs组和PBS组的GFAP的荧光表达差异没有有统计学意义。
3.4Shh-BMSCs增加了大鼠脊髓损伤后脊髓前角运动神经元的数量
大鼠脊髓损伤治疗28日，通过尼氏染色检测脊髓组织中脊髓前角运动神经元的数量。同BMSCs组和PBS组相比，Shh-BMSCs组脊髓前角运动神经元数量明显增多，BMSCs组脊髓前角运动神经元数量多于PBS组，差异有统计学意义。
3.5Shh-BMSCs减少了大鼠脊髓损伤后空洞的面积
大鼠脊髓损伤治疗28日，通过HE染色检测脊髓损伤后空洞面积的变化。同BMSCs组和PBS组相比，Shh-BMSCs组脊髓损伤空洞面积明显缩小，BMSCs组脊髓空洞面积小于PBS组，差异有统计学意义。
4.Shh-BMSCs促进运动功能的康复
在BBB评分期间，从2周到6周，Shh-BMSCs组大鼠的BBB评分明显高于BMSCs组和PBS组，BMSCs组明显高于PBS组，差异有统计学意义。
小结
Shh-BMSCs在治疗脊髓损伤的过程中，Shh可以持续分泌，Shh表达增强，促进了BMSCs的存活，增强了神经的保护和运动功能的康复，能够促进脊髓损伤后神经功能的恢复，可以为临床治疗脊髓损伤提供更好的方法。
Spinal cord injury (SCI) is one of the problems of the medical challenge, whichdue to injury, traffic accident, landslides, natural disasters and etc. SCI, which canlead to loss of cells and damage of neural tracts, therefore result in many tissular andorganic dysfunction, is one of the most common disability and lethal disease. Patientsafter SCI will not only bring serious physical and mental damage, but also cause ahuge economic burden to the whole society, therefore seriously affect the quality ofhuman life.
At present the main treatment of spinal cord injury include surgery removingoppression, steroids, protein kinase, metalloproteinases, suppression and regenerationtechnology and etc, however the effect of treatment is limited. In recent years, how torepair spinal cord injury has achieved great progress. Stem cell gene transplantation isrecognized as a promising treatment, and is the focus for the treatment of spinal cordinjury.
Previous studies have shown that bone marrow mesenchymal stem cells(BMSCs), which have widely source, a good ability of proliferation anddifferentiation, easy to obtain and cultivation, low immune rejection, and don’tinvolve the ethical issues, are the ideal carrier of the gene therapy. In their treatmentfor spinal cord injury, BMSCs can secrete neurotrophic factors, improve themicroenvironment of the spinal cord injury, differentiate into neurons which replacenecrosis or death of nerve cells, have the bridge role which can provide the axonregeneration with a good connection channel, promote the formation of new blood vessels and target spinal cord lesions. However, most BMSCs die aftertransplantation as a result of ischemia and hypoxia in the region of SCI.
Growth factor is essential for repairing spinal cord injury. Sonic hedgehog (Shh),which is produced by the notochord and floor plate, is a member of the family ofhedgehog proteins and is essential for the development and differentiation of thenervous system. Shh works on several aspects of spinal cord regeneration: it inducesthe production of motor neurons and oligodendrocytes, enhances the delivery ofneurotrophic factor to improve the microenvironment in BMSCs, facilitates neuronalsurvival, promotes axonal growth, and prevents activation of the astrocyte lineageafter SCI. However, an injection of Shh protein after SCI does not have a good effecton functional behavioral recovery because of the fast clearance of Shh protein fromthe spinal cord.
In our study, we speculated that transplantation of BMSCs transfected with Shh(Shh-BMSCs), which can provide long-term stable expression and secretion of highlevels of Shh, could conquer the difficulties of inherent in treatment with Shh alone,to facilitate repair and recovery after SCI.
In this study we referred to cell and animal research. The research mainly coversthe following three parts:
1. To isolate, culture and identification of rat BMSCs
2. To construct Shh carrier, use the lentiviral vector technology to transfect BMSCsand identify the transduction effect.
3To establish rat spinal cord injury model, treat SCI using Shh-BMSCs via thesubarachnoid injection and observe the therapeutic effects after SCI.
Section one Isolation, culture and identification of rat bonemarrow mesenchymal stem cells
Objective
To establish a method of isolation, cultivation of rat bone marrow mesenchymalstem cells (BMSCs) in vitro, to identify cell morphology, cell proliferation, cellsurface markers, osteogenetic and adipogenic differentiation capacity.
Research methods
1.Animals
Adult female Sprague Dawley (SD) rats,160-200g, were purchased fromAnimal Center of Chinese People’s Army Military Medical and Scientific Academy,Beijing, China.
2. Isolation of BMSCs and cultivation of BMSCs in primary
culture
Animals were anesthetized with10%chloral hydrate. Take the rat femur andtibia rapidly and wash them using PBS. Use scissor to cut off the epiphyseal end,expose the long bone marrow cavity, and wash them using L-DMEM mediumcontaining100U/ml penicillin streptomycin until the marrow cavity was white. Thesingle cell suspension was centrifuged by1200r/min for5minutes and cultured bySD MSC complete medium at37°C under a5%CO2atmosphere. The inoculationconcentration is6-8×l06/ml.
3. The cultivation, purification and passage of BMSCs
In the process of primitive culture, change a half quantity medium after48hoursand follow a full volume replacement every three days. When BMSCs were coveredabout80%, use trypsin-EDTA solution to digest them. The ratio of subculture was1:3.
4. The cell morphology of BMSCs
To observe the cell morphology and growth condition using inverted microscopeand photograph them.
5. To detect cell proliferation using MTT methods
At passage3, BMSCs were cultured in96-well culture plate under a5%CO2atmosphere. The inoculation density was5x103/hole. Take out one plate and detectit every day by MTT method. Detect the optical density of palte for seven days usingenzyme-linked immune detector absorption value, and the wavelength was490nm.The observation time was for the horizontal axis and the value of optical density wasthe vertical axis.
6. To detect the cell surface markers of BMSCs using flowcytometric analysis
At passage3, BMSCs were trypsinized and centrifuged by1200r/min for5minutes at4°C. Count the cells and join the monoclonal antibody CD29, CD34,CD44, and CD45for30min at room temperature. After washing cells using PBS toremove the uncombined antibody, join the second antibody labeling FITC and PE in adark place for30min, and detect them using flow cytometric analysis.
7. Osteogenetic and adipogenic differentiation capacity ofBMSCs
At passage3, BMSCs were cultured in6-well culture plate. When BMSCs werecovered about80%, join the osteogenic and adipogenic media and change a fullvolume replacement every three days, at the same time set up a blank control withoutjoining induction media. After cells was fixed by40g/L paraformaldehyde at roomtemperature for10minutes, we evaluated osteogenic differentiation by alizarin redstaining at day28and the adipogenic differentiation was assessed at day23bystaining with oil red-O solution. Observe and photograph them using inverted microscope.
Results
1. The morphological observation of BMSCs
BMSCs were spindle and showed radial colony arrangement. Cells kept goodgrowth and could passage in continuous over10passages.
2. The cell growth curve of BMSCs
Cell growth curve demonstrated that BMSCs were consistent with the growthcharacteristics and strong activity of normal cells.
3. The expression of BMSCs cell surface markers
At passage3, BMSCs were found to express cell markers CD29and CD44, butCD34and CD45were not detected.
4. The identification of osteogenic and adipogenicdifferentiation
After osteogenic and adipogenic differentiation, alizarin red and oil red-Ostaining were positive.
Summary
The whole bone marrow adherence method is simple, and can isolate, purify andamplify BMSCs in vitro. The obtained cells have general biologicalcharacteristics ofbone mesenchymal stem cells, and also have potentiality of osteogenic andadipogenic differentiation. This experimental way has important practicalsignificance to provide suffient source of carrier cells for gene engineering.
Section two Construction of bone marrow-derivedmesenchymal stem cells expressing the Shh transgene andidentification
Objective
To construct the Shh gene modified BMSCs using a lentiviral vector in vitro andthen observe the identification of transduction efficiency, provide a potential stablestem cell source for the treatment of SCI.
Research methods
1. Construction of Shh carrier
Firstly amplify attB1-Shh-attB2using overlapping PCR; then constructpDown-Shh using Gateway Technology; finally structure pLV.EX3d.P/puro-EF1α>Shh>IRES/DsRed(red fluorescent protein)Express2using Gateway Technology.
2. Constuction of lentiviral vector and BMSCs expressing theShh transgene using them
Transfection of293T cells using Lipofectamine2000. Detect the titers oflentivirus using hole-by-hole dilution method. Infect BMSCs with shh lentiviralvector and detect the transfection efficiency through the fluorescence.
3. Detect the shh mRNAexpression of Shh-BMSCs usingReal-time PCR analysis
Total RNA was extracted from the cells. Total RNA was reverse transcribed intocDNA under a PCR-reaction system and the condition of Reverse transcription.Record the value of Ct, amplification curve and dissolve curve. Calculate theexperimental results (2-ΔΔCT) using relative quantitative methods.
4. Detect the shh proteinn expression of Shh-BMSCs usingWestern blot analysis
After the proteins were collected from cells, protein concentrations were testedby the BCA method. Configuration of12%SDS-polyacrylamide gel, on sample,electrophoresis, transfer the membrane, blocked, reaction of primary and secondaryantibody, electrochemiluminescence detection.
Results
1. Construction of BMSCs expressing the Shh transgene
The shh lentiviral vector was constructed. It was entirely correct by enzymedigestion and sequencing identification, and it could transfect293T cells and achieveexpression. The lentiviral titer was2-4×108TU/ml. The fluorescence expressionincreased significantly at5d following transduction
2. Shh-BMSCs increased the shh expression
Shh-BMSCs could long-term stable mRNA and protein expression of high levelsof Shh using lentiviral vector comparing with the uninfected BMSCs.
Summary
Construction of Shh lentiviral vector successfully and Shh-BMSCs, which canlong-term stable expression of high levels of Shh. BMSCs were the basis for thesubsequent experiments.
Section three Effect of bone marrow-derived mesenchymalstem cells expressing the Shh transgene on recovery after spinalcord injury in rats
Objective
To establish the spinal cord injury model and observe the BMSCs survival andfunctional recovery via transplantation of Shh-BMSCs at7days after SCI.
Research methods
1.Animals
Adult female Sprague Dawley (SD) rats,200-220g, were purchased fromAnimal Center of Chinese People’s Army Military Medical and Scientific Academy,Beijing, China.
2. Spinal cord injury model
Animals were anesthetized with10%chloral hydrate (0.3ml/100g,intraperitoneal injection); then followed by Allen’s methods. In brief, the spinal cordwas exposed by performing T10laminectomy. SCI was induced by dropping a10-grod (diameter,2.5mm) from a height of25mm onto an impounder positioned on thespinal cord.
3. Treatment of Shh-BMSCs after SCI via intrathecal injection
Seven days after SCI, a second laminectomy was performed at lumbar level4.We therefore microinjected Shh-BMSCs into the model rats intrathecally. In thecontrol group, BMSCs or PBS was injected.
4. Detect the shh protein expression after treatment ofShh-BMSCs in rats with SCI
To detect the shh protein expression using Western blot analysis at28d aftertreatment.
5. Detect the neuroprotection after treatment of Shh-BMSCs inrats with SCI
5.1The expression of neurotrophic factors
To detect the basic fibroblast growth factor (bFGF) and vascular endothelialgrowth factor (VEGF) protein expression using Western blot analysis at28d aftertreatment.
5.2Shh-BMSCs promoted the BMSCs survival
To observe the lesion epicenter using a fluorescence microscope at28d aftertreatment and analyze the number of BMSCs using Image J software.
5.3Detection of neurofilament200(NF200) and glial fibrillaryacidic protein (GFAP) immunofluorescence staining
The sections around the lesion epicenter were washed, blocked, incubated byprimary antibodies (NF200and GFAP) and secondary antibody and stained by DAPIat28d after treatment. The expressions of GFAP and NF200were observed using afluorescence microscope and analyzed by Image J software.
5.4Nissl’s staining for the motoneurons
The sections around the lesion epicenter were stained in1%solvent blue at60°Cfor40min, washed three times using distilled water, differentiated in95%ethanol,dehydrated in100%ethanol,cleared in xylene and covered by using a resinousmedium. Photograph and count the motoneurons using a inverted microscope.Analyze them by Image J software.
5.5Evaluate the lesion cavity using HE staining
The sections around the lesion epicenter were stained in Harris hematoxylin for7min and0.5%eosin for2-4min. The volumes of lesion cavity were photographedusing a fluorescence microscope and analyzed by Image J software.
6. Evaluate the behavioral recovery after treatment ofShh-BMSCs in rats with SCI
The Basso–Beattie–Bresnahan (BBB) locomotor rating score is widely used toevaluate hind-limb motor function. Rats were examined by three trained examinersfor6weeks after SCI in a double-blinded manner. The BBB results were averaged.
7. Statistical analysis
All data were expressed as mean±SD. One-way analysis of variance (ANOVA)was used to compare mean values. A repeated measure ANOVA was used to analyzeweekly BBB scores in different groups. Statistical analyses were performed usingSPSS15.0software. Statistical evaluations were considered significant at P <0.05.
Results
1. The model of spinal cord injury was successfully established
Rat tail swing and hind limb extremity paralysis indicated that induction of SCIwas successful. Manual bladder expression was performed before recovery ofautonomous urination. All rats were injected penicillin intraperitoneally and survivedbetter.
2. Shh-BMSCs promoted the expression of Shh in vivo
Shh expressions of spinal cord were detected by Western blot analysis at28dafter treatment. The protein expression of Shh was significantly greater afterShh-BMSCs treatment than the other two interruptions. Differences were consideredstatistically significant (P <0.05).
3. Shh-BMSCs increased the neuroprotection
3.1Shh-BMSCs promoted the expression of bFGF and VEGF
The bFGF and VEGF protein levels were determined using western blot analysisat day28after treatment.The protein levels of bFGF and VEGF in the Shh-BMSCsgroup were markedly higher than those in the PBS and BMSCs groups, and the bFGFand VEGF protein expression in the BMSCs group increased significantly by day28in comparison with the levels in the PBS group. Differences were consideredstatistically significant (P <0.05).
3.2Shh-BMSCs enhanced the BMSCs survival
BMSCs survival was detected using a fluorescence microscope at28d aftertreatment. The number of BMSCs present at this time point in the Shh-BMSCs groupwas significantly more than those in the BMSCs group on day28after transplantation.Differences were considered statistically significant (P <0.05).
3.3Shh-BMSCs increased the expression of NF200and reducedthe expression of GFAP
The expressions of NF200and GFAP were detected using immunofluorescencestaining. On day28after transplantation, the expression of NF200in the Shh-BMSCsgroup was significantly higher than that in the other two groups, and the staining inthe PBS group was significantly lower than that in the BMSCs group. The expressionof GFAP on day28after transplantation was markedly weaker in the Shh-BMSCsgroup versus the other two groups, and the staining between the PBS and BMSCsgroups did not differ significantly. Differences were considered statisticallysignificant (P <0.05).
3.4Shh-BMSCs increased the number of motoneurons after SCI
Nissl’s staining at the ventral horns was used to evaluate the number ofmotoneurons at28d after treatment. Injection of Shh-BMSCs significantly increasedthe number of motoneurons in the ventral horns, compared with injection of BMSCsor PBS. The BMSCs group had more motoneurons than the PBS group at the ventralhorns. Differences were considered statistically significant (P <0.05).
3.5Shh-BMSCs reduced the area of lesion cavity after SCI
HE staining was used to evaluate the area of lesion cavityat28d after treatment.Injection of Shh-BMSCs significantly reduced the area of lesion cavity, comparedwith injection of BMSCs or PBS. The BMSCs group had less area of lesion cavitythan the PBS group. Differences were considered statistically significant (P <0.05).
4. Shh-BMSCs promoted significant functional recovery
BBB tests showed highly significant motor improvement in rats that had beentreated with Shh-BMSCs compared with those treated with PBS or BMSCs, from2wto6w after SCI. The BBB score of the BMSCs group was significantly greater thanthose of the PBS group (P <0.05).Differences were considered statisticallysignificant.
Summary
BMSCs that have been transfected with a Shh transgene, which can providelong-term stable expression and secretion of high levels of Shh and so enhance thesurvival of BMSCs, would promote the neuroprotection and facilitate repair andrecovery after SCI and could be a potential valuable therapeutic intervention for SCIclinically.
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