构建OECs-VEGF165基因工程细胞的实验研究
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摘要
研究背景
(?)脑白质疏松症的高发生率与治疗滞后
随着人口老龄化的日益加剧、人们保健意识的增强以及影像学技术的发展,脑白质疏松症(Leukoaraiosis, LA)也备受关注,它是老年人认知障碍最常见的原因之一,正常老年人的发病率为1.7-8.6%,高血压患者为78%-93%, Alzheimer氏病为30%-31%,脑卒中病人为36%-100%。LADIS (Leukoaraiosis and Disability in the Elderly)研究发现：在没有残疾的老年人中,机体能力的水平和脑白质损害的程度相关,LA白质损害的程度和痴呆残疾评估分数密切相关。目前多数学者认为LA是由于年龄、高血压及其他血管病危险因素造成脑内小动脉发生结构变化,最终导致的脑部缺血性损伤。高血压、老龄的LA的最主要危险因素。
LA作为一种慢性血管性缺血性病症,脑缺血后的神经发生和血管发生是由缺血引发的脑内两个主要病理生理过程,两者在促进缺血性损伤后的神经功能恢复方面都有重要作用。只有从改善局部的血管供血,才能从根本上解决LA发生发展的病因,阻止其向更严重(痴呆、脑梗塞)的病情发展。
(?)血管内皮生长因子的研究进展和分析
治疗性血管再生(therapeutic angiogenesis)是近年提出的一个新概念,率先在心肌缺血方面被重点研究,指刺激心肌缺血区的小血管生长和促进侧枝循环形成,即心肌缺血区的“自我搭桥”。目前认为,血管内皮生长因子(vascular endothelial growth factor, VEGF)是最有效的刺激血管生长的细胞因子之一,VEGF的生物学功能和可使新生血管形成的特性己引起越来越多人的关注,国外有人把VEGF基因治疗称为“分子搭桥术”,有望部分取代外科搭桥术和血管成形术。有研究发现,脑缺血后VEGF及其受体的表达上调,脑内注射VEGF导致脑部新生血管的形成和VEGFR-1在星形细胞的表达,VEGF还可直接发挥神经保护作用而不依赖血管形成,有助于皮质神经元免受低氧和低糖的损害,刺激轴突的生长。
有关LA病变内VEGF变化及对LA的治疗国内外未见报道。VEGF在心脑血管方面的成功实验,为我们对LA的研究打下坚实的基础。如何应用外源性VEGF蛋白及VEGF基因治疗脑血管病,改善受损脑组织的血液供应,减轻其缺血程度,尚需进一步探索。LA的病理改变主要表现为缺血性脱髓鞘、髓鞘含量下降,如果能使局部的髓鞘再生,或将控制LA的进展,改善临床症状。
嗅鞘细胞的生物学特性
嗅鞘细胞(Olfactory ensheathing cells, OECs)作为一种具有星形胶质细胞和雪旺细胞双重特性的细胞,保留了许多发育阶段的特性和可塑性,OECs的存在使嗅系统成为迄今发现唯一可以再生的中枢,它的生物学功能极其广泛。对神经元的生长、发育、存活以及对突起生长有明显的促进作用。Ramon-Cueto等研究表明啮齿类动物的OECs能够使脊髓损伤大鼠轴突再生并使再生的神经纤维和脱髓鞘的神经纤维复髓鞘,因而,OECs为细胞移植治疗CNS疾病开辟了一条希望之路。
基于OECs的生物学作用,目前的许多研究都集中在应用OECs移植来治疗神经组织的脱髓鞘损伤。通过对OECs与凋亡关系的研究,可以加强对OECs治疗作用和机制的理解,扩展其治疗范围和改进其治疗方法。
》建立OECs-VEGF基因工程细胞的构想和意义
随着基因工程技术研究的不断深入,基因治疗成为脑血管疾病防治新的切入点,基因转染技术使利用两者治疗LA成为可能,建立基因工程细胞的构想随之产生。
基于以上分析,设想：利用嗅球OECs进行培养,诱导血管内皮再生的VEGF目的基因转入受体细胞,构建高度表达VEGF的大鼠OECs-VEGF基因工程细胞株,将移植细胞注入脑内,使其不但可以充分发挥两者的协同作用,并为解决VEGF表达量较少、持续性欠佳这一瓶颈问题提供了有效的解决途径。从而为LA的细胞移植治疗和基因治疗探索出一种新的切实可行的具有突破性的方法。预期OECs在LA模型的脑内大量传代扩增,向神经系细胞分化,具有促进轴突再生和修复髓鞘的作用；同时,经过转染的OECs生物活性更强,持续分泌更多的促进血管内皮再生的VEGF,从而改善LA神经组织的微循环,促进血管再生,减轻神经组织的缺血性损伤。
本研究旨在嗅球OECs的分离、培养、纯化,携带human-VEGF165基因慢病毒的构建、合成以及病毒对OECs的转染,完成OECs-VEGF基因工程细胞的构建。
研究目的：
1.探索将human-VEGF165基因导入OECs的方法,分析构建OECs-VEGF基因工程细胞的可行性。
2.研究从大鼠嗅球分离培养纯化OECs的方法与技巧。
3.构建携带human-VEGF165基因的慢病毒载体。
4. human-VEGF165-LV转染OECs,构建OECs-VEGF165基因工程细胞。
研究方法：
1.嗅球OECs的取材、分离、培养、纯化与鉴定。
将雄性SD大鼠断头处死后,取嗅球嗅神经层和嗅球颗粒层剪碎,放在胰酶中,置培养箱内消化。用含FBS的DF12终止消化,吹打,静置,吸取上层悬液待接种,重复3次。OECs的纯化方法主要为差速贴壁法。将细胞悬液接种,加入双抗,培养。调整细胞,种植,贴壁。每3天换液一次。取原代细胞,制成细胞悬液。在24孔板中接种,测细胞总数,求平均值,持续计数9天,做出生长曲线。
采用免疫荧光鉴定OECs原代细胞纯度：吸去培养液,反复漂洗、固定、孵育细胞两次,滴加Hoechst33342复染核,滴加碱性甘油于载玻片上,p75染色,封固后荧光显微镜下观察。计算高倍镜下视野中核的数目以及阳性细胞数,并计算阳性细胞数与核数的比例,即为OECs的纯度。
2.构建携带human-VEGF165基因的慢病毒载体。
使用慢病毒包装试剂盒,通过三种质粒的共同转染293T细胞,合成携带目的基因的慢病毒载体。
1)克隆构建：①过表达目的基因的载体制备：利用PCR方法钓取目的基因,将目的载体进行酶切,交换,其产物转化细菌感受态细胞。②明确目的基因及载体信息。③DNA琼脂糖凝胶电泳。④用CaC12制备新鲜的大肠杆菌感受态细胞。⑤转化：将已转化的感受态细胞转移到琼脂培养基上。长出的克隆进行后续PCR鉴定。
2)质粒表达检测：①准备目的细胞。②目的细胞质粒转染：制成细胞悬液,接种于培养板中,根据Lipofectamine2000转染试剂使用说明书进行转染操作。转染后观察质粒上荧光标记基因的表达情况以判断感染效率。③蛋白印迹法检测目的基因表达,将收集的细胞提取蛋白进行蛋白印迹检测,同时提取7860细胞,和空质粒转染的293T细胞分别作为空白对照和阴性对照。
3)病毒包装和收集浓缩：细胞状态对于病毒包装至关重要,因此需要保证良好的细胞状态和较少的传代次数。收集浓缩后,进行病毒生物学滴度测定。
4)病毒滴度检测：使用荧光实时定量PCR法测定病毒滴度,通过比较control组和实验组的Ct值差异判断滴度值。
3. human-VEGF165-LV转染OECs、构建OECs-VEGF165基因工程细胞。
1)预实验：分四组进行,每组四个不同梯度的MOI (Multiply of Infection),共16孔,其中每种实验条件做三个复孔。每组均有不同梯度的MOI值。
2)免疫荧光检测：将未转染的OECs与转染后OECs传代接种,弃培养液,漂洗细胞两次,室温固定,PBS洗涤,加入正常山羊血清后进行孵育,洗涤,再在爬片上滴加Hoechst33342复染核,碱性甘油封固后荧光显微镜下观察。同时在染色过程中制作有阴性对照玻片,未加一抗。
3)Real time-PCR:①总RNA提取：取6孔板细胞,用吹打管吹至液体澄清,无细胞团块转至EP管中。用力摇晃试管、离心。用移液枪转上层水相于另EP管中,加等体积异丙醇,沉淀RNA,离心,用紫外光度检测RNA浓度。②RT-PCR:进行转染后OECs与未转染OECs细胞总RNA的逆转录。RT试剂盒中试剂解冻后,摇晃试管使其均匀,再稍离心,放于冰盒上操作。③Real-time PCR,融解曲线分析。
4)ELISA检测：在标准品孔及待测样本孔中分别加入酶联亲和物,包被微孔板覆膜、孵育后扣干孔内剩余残液,每孔依照次序分别加入底物,避光反应后每孔分别加入终止液,终止反应。使用酶标仪读取OD值。Logit-Log直线回归计算、绘制出标准曲线图。
研究结果：
1.嗅球OECs的取材、分离、培养、纯化与鉴定。
1)差速贴壁法所取出的原代细胞约在所有细胞中占98%,但是随着代数的增加,OECs的纯度很快下降,在第二代约占78%,到第三代纯度降至58%。
2)细胞形态和生长特性：纯化后OECs形态较为一致,主要呈梭性,约占细胞总数的90%以上；OECs也有其它形态,比较常见的有多突起型和不规则型。传代后OECs的形态发生改变,增殖形成的紧密连接消失,细胞也从梭性变成多突起形状。在培养液营养物质充分且完全的情况下,活性差的细胞渐渐凋亡,脱离瓶底,但对于大多数的细胞则可从多突起形态转化为增殖能力较强的梭形形态,继续增殖。
3)原代OECs的增殖能力强,但随着传代次数的增加,OECs纯度下降,活性也明显下降,并且细胞的形态变得多种多样,增殖变缓。
2.构建携带human-VEGF165基因的慢病毒。
1)质粒克隆载体的构建：从载有humanVEGF165基因的质粒上扩增出目的基因片段,并重新链接与用于慢病毒包装载体的质粒上,通过对连接上的阳性转化子测序,并与NCBI上的目的基因序列进行比对,得出包装质粒上连接的目的基因序列正确。
2)质粒表达目的基因检测：携带有EGFP蛋白基因做标记的质粒通过脂质体转染入293T细胞内,24小时后细胞内即可观察到明显的荧光,表明目的基因与EGFP表达正常。目的基因蛋白预测大小为22KD。通过Western Blot检测,从质粒转染成功的293T细胞提取的蛋白中可以检测到22KD附近处有条带,其大小和目的基因合成的蛋白分子量相吻合。这表明连接于克隆载体质粒上的目的基因在进入细胞后过表达成功。
3)合成病毒的滴度：通过Real-time PCR来检测病毒的滴度,得出在相当于病毒原液万分之一组(即梯度稀释中的10-4ul组)和Con组样品的Ct值出现2个循环左右差异,认为在10-4ul组样品中存在病毒颗粒,得出病毒滴度为2×108个／ml。
3. human-VEGF165-LV转染OECs、构建OECs-VEGF165基因工程细胞1)通过对不同浓度的病毒,以及不同转染条件的比较,得出对OECs慢病毒基因载体的最佳感染条件如下：对于合成的慢病毒,在MOI为10时即得到大于80%的感染效率；相对于普通培养基,Eni.s培养基并不能明显提高感染效率；Polybrene同样对病毒的感染效率改变不大。
2)转染后,OECs的生长活性受到影响。12小时内进入“休止期”,24小时后,细胞培养上清内死细胞增多,荧光不可见,48小时后,转染组的细胞死亡的更多,72小时后,少量细胞可见微弱荧光表达,96小时后,大部分细胞内出现EGFP的表达。
3)转染组MOI三个不同亚组,GFP阳性细胞数占总细胞数的比例大于80%。但是在转染后,10与20亚组细胞死亡较多,MOI为5亚组细胞死亡较少,细胞功能恢复快。对转染成功的OECs进行VEGFA免疫荧光染色,可观察外源VEGFA的表达。通过对转染效率,转染对OECs生长的影响,初步确定大量转染OECs时,MOI值为5是合适的选择。
结论
1.通过差速贴壁法纯化从嗅球提取出的OECs有高纯度和较好的活性,是体外研究OECs时提取和纯化原代细胞简便、经济的方法,适合进行相关的进一步研究。
2.随着原代细胞传代次数的增加,OECs的纯度和活性都明显下降,因而在OECs研究时,要选择合适传代次数下的OECs进行研究,使结果具有代表性。
3.携带有human-VEGF基因的慢病毒载体可以成功将目的基因转入OECs,并且由于慢病毒对原代细胞感染的高效性,在尽量小的影响OECs活性的情况下,达到较高的转染效率。
4.通过携带有human-VEGF基因的慢病毒载体感染OECs,构建出的human-VEGF-OECs基因工程细胞可稳定、高效表达VEGF165基因,合成的目的蛋白分泌到细胞外,符合VEGF165蛋白的生理特点,完成其在正常机体中的生物学作用。
Background
(?) High incidence and lagging treatment of leukoaraiosis
With the growing aging of population, the reinforcement of people's awareness about health and the development of imaging technology, leukoaraiosis (LA) has been attracting much attention. It is one of the most common cause of cognitive disorders of the elderly, in comparison with the incidence rate of1.7-8.6%of healthy elderly,78%-93%of the elderly patients with hypertension,30%-31%of the elderly patients with Alzheimer's disease and36%-100%of the elderly patients with stroke. The LADIS (Leukoaraiosis and Disability in the Elderly) study found that the body's ability level was related with the extent of white matter damage and the degree of white matter lesions was closely correlated with dementia disability assessment scores in the elderly not suffering from disability. Currently, most scholars believe that LA is due to the brain artery structural changes induced by age, hypertension and other vascular risk factors, which eventually leads to the brain ischemic injury. Hypertension and aging are the most important risk factors for LA.
LA is a chronic ischemic disease. After cerebral ischemia neurogenesis and angiogenesis are triggered as two major pathophysiological processes, which have an important role on promoting nerve function recovery after ischemic injuries. To improve the local blood supply is a fundamental solution to LA development, preventing the progression of the disease to the more serious (dementia, cerebral infarction).
(?) Research progress and analysis of vascular endothelial growth factor
Therapeutic angiogenesis is a new concept in recent years. It is the first to study on myocardial ischemia, aiming to stimulate the growth of the small blood vessels and to promote collateral circulation of the myocardium ischemic area, namely "self-bypass"."Currently, vascular endothelial growth factor (VEGF) is one of the most effective cytokines to stimulate the growth of blood vessels. The biological functions and characteristics of VEGF have attracted more and more attention. VEGF gene therapy is viewed as "molecular bypass surgery", which is expected to partially replace surgical bypass grafting and angioplasty. Study found that VEGF and its receptors upregulated after cerebral ischemia, intracerebral injection of VEGF led to the formation of new blood vessels in the brain and VEGFR-1expression in astrocytes and that VEGF also directly played a neuroprotective effect without relying angiogenesis, which contributed to the damage of cortical neurons from hypoxia and low sugar and stimulated the growth of axons.
There are rare reports about VEGF changes in the lesions and treatment of LA at home and abroad. The success of VEGF in the heart cerebrovascular experiments lay a solid foundation for the study of LA.It needs the further explorations about how to apply exogenous VEGF protein and VEGF gene therapy on cerebrovascular disease, improve the blood supply to the damaged brain tissue and reduce the extent of their ischemic, LA pathological changes mainly are ischemic demyelination and the decline in myelin content. Partial remyelination can control the progress of the LA and improve the clinical symptoms.
(?) Biological characteristics of olfactory ensheathing cells
Olfactory ensheathing cells (OECs) are cells of the dual characteristics of the astrocytes and Schwann cells and retain many of the characteristics of the developmental stages and plasticity. The olfactory system is the only renewable hub so far and its biological functions are extremely wide, exerting distinct facilitation on the growth, development and survival of neuron as well as promoting neurite outgrowth. Ramon-Cueto, studies have shown that rodents OECs can accelerate axonal regeneration and the remyelination of the demyelinated nerve fibers in rats with spinal cord injury. Therefore, OECs has opened up a new hope for cell transplantation in the treatment of CNS disorders.
Based on the biological role of OECs, many studies have focused on the application of olfactory ensheathing cell transplantation to treat demyelination of nerve tissue. Based on the study about the relationship between OECs and apoptosis, it can enhance the understanding of the therapeutic effect and mechanism of OECs and expand its range of treatment and improve their treatment.
(?) Composition and significance of the establishment of the genetically engineered cells OECs-VEGF
With the deepening of the study of the mechanism of the molecular level, gene therapy has become the new entry point for cerebrovascular disease prevention and treatment. Gene transfection technology enables the use of both treatment possible and the idea of composing genetically engineered cells is consequently established.
Based on the above analysis, it is envisaged that OECs are cultured. VEGF target gene is transferred to the recipient cell and builds OECs-VEGF gene engineering cells of rats with high expression of VEGF. The transplanted cells are injected into the brain. They not only can exert the synergic effect, but also resolve the low expression and poor continuity of VEGF which is the bottleneck problem to provide effective solution. LA cell transplantation and gene therapy to explore a new kind of practical breakthrough. Expected OECs are passaged and amplified in large quantities in the the LA model, and eventually differentiated to the nervous system, which has a role to promote axonal regeneration and repair of myelin. Meanwhile, after transfection the OECs have stronger biological activity and continually secret more VEGF to promote vascular endothelial regeneration, thereby improving the LA nerve tissue microcirculation, promoting angiogenesis, and reducing ischemic injury of the nerve tissue.
The purpose of this study was to separate, culture and purify OECs from the olfactory bulb. In addition, the lentivirus carrying human-VEGF165gene is construct and transfected OECs. Then OECs-VEGF gene engineered cells are established.
Objectives
1. To explore the method of introducing human-VEGF165gene into OECs and analysis the feasibility of building the OECs-VEGF genetically engineered cells.
2. To research the technique of purifying OECs from the olfactory bulb isolated rat.
3. To construct the lentivirus vector carrying the human-VEGF165gene
4. To building the OECs-VEGF165genetically engineered cells by human-VEGF165-LV transfecting OECs
Methods
1. Sampling, separation, cultivation, purification and identification of OECs from olfactory bulb
Male SD rats were decapitated. The olfactory nerve layer and granular layer of the olfactory bulb were taken, cut into pieces and digested in trypsin in the incubator. DF12containing FBS terminated the digestion. It was blown and beaten. Draw the upper suspension to be inoculated after standing a while and repeat three times. The OECs were mainly purified by differential adherence method. Cell suspension was joined with dual antibody and cultivated for2d so ld.The cultivated cells were adjusted, planted and adhered. Medium was changed every3days. Take the primary cells, prepare a cell suspension which was inoculated in24-well plates, count the total number of cells, average for the following nine days and make the growth curve.
The purity of primary OECs is identified by immunofluorescence. Remove the culture medium, rinse repeatedly, fix and incubate twice. The nuclear was stained with Hoechst33342. Alkaline glycerol was dropped on glass slides. After p75staining, sealed and observed with fluorescence microscopy. Counted the number of nuclear and positive cells in high magnification view and calculated the ratio of the positive cells, which is the purity of OECs.
2. Construct the lentivirus carrying the human-VEGF165gene
A lentiviral vector carrying the desired gene was constructed by three plasmid co-transfecting of293T cells with the lentivirus packaging kit.
1) Cloning Construction:①The carrier preparation which overexpressed objective gene:catching the target gene by PCR, digesting the vector and exchange and transforming its product into competent cells of bacteria.②Making the information about target gene and vector clear.③DNA agarose gel electrophoresis.④Preparing fresh E. coli competent cells by using the CaCl2.⑤Transferring the transformed competent cells to the agar medium. Grown clones were identified by subsequent PCR.
2) Plasmid expression detection:①Preparation of the purpose cells.②Target cell plasmid transfection:preparing a cell suspension, seeding in culture plates and transfecting according to the Lipofectamine2000transfection reagent manual operation. In order to determine the efficiency of infection was judged by fluorescently labeled plasmid gene expression.③Proteins were extracted from the collected cells and were detected by western blotting, Meanwhile,7860cells and empty plasmid-transfected293T cells were used as blank control and the negative control.
3) Viral packaging, collecting and concentrating:the state of cells was essential for viral packaging, so we needed ensure a good state of cells and less number of passages. After collecting and concentrating, viral biological titer was determinated.
4) Viral titer detection:the titer was detected by fluorescence real-time quantitative PCR and determined by comparing the Ct value differences between the control group and the test group.
3. Building the OECs-VEGF165genetically engineered cells by human-VEGF165-LV transfecting OECs
1) Pre-experiment:four groups, each of which had four different gradients MOI (Multiply of Infection), a total of16holes, there were three wells for every experimental condition. MOI value in each group had a different gradient.
2) Immunofluorescence detection:After inoculation and passage untransfected and transfected OECs, discard the culture medium, rinse the cells twice, fix twice at room temperature, wash3times with PBS and incubate after addition of normal goat serum. Then wash, stain nucleus in climbing slices with Hoechst33342, seal with alkaline glycerol and observe under fluorescence microscopy. At the same time, the slides without adding antibody in the dyeing process were used as the negative control slides.
3) Real time-PCR:①Extraction of total RNA:blow the cells in6-well plates until they were clear then transfer them to EP tubes. Firmly shake and centrifuge the test tubes. By pipette transfer upper aqueous phase to another EP tube, add an equal volume of isopropanol to precipitate the RNA, centrifuge, and detectRNA concentration with ultraviolet degrees.②RT-PCR:Total RNA of the untransfected and transfected OECs was transcribed reversely after transfection. After thawing the reagents of RT kit, shake up the test tube, slightly centrifugate and perform on ice.③Real-time PCR. Melting curve analysis.
4) ELISA detection:enzyme-linked pro and material were added to the standard holes and test sample hole. After microplate coated and drained after incubation, the substrate was added to each well in accordance with the order respectively. After kept in the dark, the liquid to terminate the reaction was added. OD value was shown with a microplate reader. Calculated with Logit-Log linear regression and draw a standard graph.
Results
1. Sampling, separation, cultivation, purification and identification of OECs from olfactory bulb
1) The primary cells cultivated by differential adherent method approximately accounted for98%of all cells. More the times of passage were, lower the purity of OECs was. In the second generation the purity of OECs declined to78%and the third was58%.
2) Cellular morphology and growth characteristics:the cellular morphology after purification was consistent, mainly spindle-shaped, accounting for about90%of the total cells. There were other common forms, multiple swelling and irregular. Cellular morphology changed, tight junctions disappeared and cells changed from spindle-shaped to multiple swelling after passage. In the case of culture liquid nutrients fully and completely, cells with poor activity died gradually and seperated from the bottom. But the most of the cells could be transformed from multiple projections to spindle-shaped form with stronger proliferation ability and continued to proliferate.
3) The proliferative capacity of primary OECs was strong. But with the times of passages increased, the purity of OECs decreased. The cell shape diversified and the proliferation slowed.
2. To construct the lentivirus carrying the human-VEGF165gene
1) Construction of plasmid cloning vector. The target gene fragment was amplified from the plasmid containing humanVEGF165gene, and re-linked to the plasmid used as lentivirus packaging vectors. By sequencing the positive transformants and comparing with the NCBI on the target gene sequences, the target gene sequence was clear.
2) Detection of plasmid expressing the gene. The plasmids labeled with the EGFP protein gene were transfected into293T cells by liposome. Significant fluorescence the cells could be observed after24hours, which indicated that the expression of target gene and EGFP was normal. The predicted size of the target gene protein was22KD. By Western Blot analysis, the protein extracted from plasmid transfected293T cells could be detected22kD near bands, which was consistent with the protein synthesized by target. This indicates that the connection was successful.
3) Synthesis of viral titer. Ct value appeared2circulating difference between one ten thousandth group (i.e. gradient group the10-4ul dilution) and Con group by real-time PCR. It was considered that there were virus particles in the samples10-4ul and virus titer of2×108/ml was obtained.
3. Building the OECs-VEGF165genetically engineered cells by human-VEGF165-LV transfecting OECs
1) From different concentrations of the virus and the comparison of different transfection conditions, the best infection conditions of lentiviral gene vector of OECs were as follows:for lentiviral synthetic, at MOI=10to obtain greater than80%of the efficiency of infectionat; relatived to the normal medium, Eni.s medium did not significantly improve the infection efficiency; Polybrene also little changed in the efficiency of virus infection.
2) After transfection, the growth activity of OECs was affected."Telogen" within12hours, after24hours, an increase in the dead cells in the cell culture supernatant which was not visible under fluorescence, and48hours more died cells in transfection group. After72hours the weak fluorescence was visible in a small amount of cells. After96hours, the majority of cells expressed EGFP.
3) GFP-positive cells accounted for more than80%in the three transfection sub-groups with different MOI. But after transfection,10and20subgroups cells died more. Cells of MOI5died less and cell function recovered quickly. VEGFA immunofluorescence staining was performed in the transfected OECs and the expression of the exogenous VEGFA was observed. From transfection efficiency and the impact of transfection on the growth of OECs, MOI=5was a more appropriate choice.
Conclusion
1. OECs from the olfactory bulb which are purified by differential adherent method appear high purity and good activity. That is a simple and economical way for extraction and purification of primary cells in vitro studies, which is suitable for further relevant studies.
2. With the times of passages increase, the purity of OECs declines significantly. Thus to make the results more representative, it is important to select the appropriate number of passages during OECs research.
3. Target gene can be transferred into OECs by lentiviral vector carrying the human-VEGF gene. Because of the high infection efficacy of lentivirus in primary cells, higher transfection efficiency can be achieved in the case of the influence of the activity of OECs as small as possible.
4. OECs are infected by the lentiviral vector carrying the human-VEGF gene to construct the human-VEGF-OECs genetically engineered cells, which can express of VEGF165gene stably and efficiently. The synthetic protein can be secreted to the extracellular which has the same physiological characteristic as VEGF165protein and can play a biological role in a normal body.
(?)脑白质疏松症的高发生率与治疗滞后
随着人口老龄化的日益加剧、人们保健意识的增强以及影像学技术的发展,脑白质疏松症(Leukoaraiosis, LA)也备受关注,它是老年人认知障碍最常见的原因之一,正常老年人的发病率为1.7-8.6%,高血压患者为78%-93%, Alzheimer氏病为30%-31%,脑卒中病人为36%-100%。LADIS (Leukoaraiosis and Disability in the Elderly)研究发现：在没有残疾的老年人中,机体能力的水平和脑白质损害的程度相关,LA白质损害的程度和痴呆残疾评估分数密切相关。目前多数学者认为LA是由于年龄、高血压及其他血管病危险因素造成脑内小动脉发生结构变化,最终导致的脑部缺血性损伤。高血压、老龄的LA的最主要危险因素。
LA作为一种慢性血管性缺血性病症,脑缺血后的神经发生和血管发生是由缺血引发的脑内两个主要病理生理过程,两者在促进缺血性损伤后的神经功能恢复方面都有重要作用。只有从改善局部的血管供血,才能从根本上解决LA发生发展的病因,阻止其向更严重(痴呆、脑梗塞)的病情发展。
(?)血管内皮生长因子的研究进展和分析
治疗性血管再生(therapeutic angiogenesis)是近年提出的一个新概念,率先在心肌缺血方面被重点研究,指刺激心肌缺血区的小血管生长和促进侧枝循环形成,即心肌缺血区的“自我搭桥”。目前认为,血管内皮生长因子(vascular endothelial growth factor, VEGF)是最有效的刺激血管生长的细胞因子之一,VEGF的生物学功能和可使新生血管形成的特性己引起越来越多人的关注,国外有人把VEGF基因治疗称为“分子搭桥术”,有望部分取代外科搭桥术和血管成形术。有研究发现,脑缺血后VEGF及其受体的表达上调,脑内注射VEGF导致脑部新生血管的形成和VEGFR-1在星形细胞的表达,VEGF还可直接发挥神经保护作用而不依赖血管形成,有助于皮质神经元免受低氧和低糖的损害,刺激轴突的生长。
有关LA病变内VEGF变化及对LA的治疗国内外未见报道。VEGF在心脑血管方面的成功实验,为我们对LA的研究打下坚实的基础。如何应用外源性VEGF蛋白及VEGF基因治疗脑血管病,改善受损脑组织的血液供应,减轻其缺血程度,尚需进一步探索。LA的病理改变主要表现为缺血性脱髓鞘、髓鞘含量下降,如果能使局部的髓鞘再生,或将控制LA的进展,改善临床症状。
嗅鞘细胞的生物学特性
嗅鞘细胞(Olfactory ensheathing cells, OECs)作为一种具有星形胶质细胞和雪旺细胞双重特性的细胞,保留了许多发育阶段的特性和可塑性,OECs的存在使嗅系统成为迄今发现唯一可以再生的中枢,它的生物学功能极其广泛。对神经元的生长、发育、存活以及对突起生长有明显的促进作用。Ramon-Cueto等研究表明啮齿类动物的OECs能够使脊髓损伤大鼠轴突再生并使再生的神经纤维和脱髓鞘的神经纤维复髓鞘,因而,OECs为细胞移植治疗CNS疾病开辟了一条希望之路。
基于OECs的生物学作用,目前的许多研究都集中在应用OECs移植来治疗神经组织的脱髓鞘损伤。通过对OECs与凋亡关系的研究,可以加强对OECs治疗作用和机制的理解,扩展其治疗范围和改进其治疗方法。
》建立OECs-VEGF基因工程细胞的构想和意义
随着基因工程技术研究的不断深入,基因治疗成为脑血管疾病防治新的切入点,基因转染技术使利用两者治疗LA成为可能,建立基因工程细胞的构想随之产生。
基于以上分析,设想：利用嗅球OECs进行培养,诱导血管内皮再生的VEGF目的基因转入受体细胞,构建高度表达VEGF的大鼠OECs-VEGF基因工程细胞株,将移植细胞注入脑内,使其不但可以充分发挥两者的协同作用,并为解决VEGF表达量较少、持续性欠佳这一瓶颈问题提供了有效的解决途径。从而为LA的细胞移植治疗和基因治疗探索出一种新的切实可行的具有突破性的方法。预期OECs在LA模型的脑内大量传代扩增,向神经系细胞分化,具有促进轴突再生和修复髓鞘的作用；同时,经过转染的OECs生物活性更强,持续分泌更多的促进血管内皮再生的VEGF,从而改善LA神经组织的微循环,促进血管再生,减轻神经组织的缺血性损伤。
本研究旨在嗅球OECs的分离、培养、纯化,携带human-VEGF165基因慢病毒的构建、合成以及病毒对OECs的转染,完成OECs-VEGF基因工程细胞的构建。
研究目的：
1.探索将human-VEGF165基因导入OECs的方法,分析构建OECs-VEGF基因工程细胞的可行性。
2.研究从大鼠嗅球分离培养纯化OECs的方法与技巧。
3.构建携带human-VEGF165基因的慢病毒载体。
4. human-VEGF165-LV转染OECs,构建OECs-VEGF165基因工程细胞。
研究方法：
1.嗅球OECs的取材、分离、培养、纯化与鉴定。
将雄性SD大鼠断头处死后,取嗅球嗅神经层和嗅球颗粒层剪碎,放在胰酶中,置培养箱内消化。用含FBS的DF12终止消化,吹打,静置,吸取上层悬液待接种,重复3次。OECs的纯化方法主要为差速贴壁法。将细胞悬液接种,加入双抗,培养。调整细胞,种植,贴壁。每3天换液一次。取原代细胞,制成细胞悬液。在24孔板中接种,测细胞总数,求平均值,持续计数9天,做出生长曲线。
采用免疫荧光鉴定OECs原代细胞纯度：吸去培养液,反复漂洗、固定、孵育细胞两次,滴加Hoechst33342复染核,滴加碱性甘油于载玻片上,p75染色,封固后荧光显微镜下观察。计算高倍镜下视野中核的数目以及阳性细胞数,并计算阳性细胞数与核数的比例,即为OECs的纯度。
2.构建携带human-VEGF165基因的慢病毒载体。
使用慢病毒包装试剂盒,通过三种质粒的共同转染293T细胞,合成携带目的基因的慢病毒载体。
1)克隆构建：①过表达目的基因的载体制备：利用PCR方法钓取目的基因,将目的载体进行酶切,交换,其产物转化细菌感受态细胞。②明确目的基因及载体信息。③DNA琼脂糖凝胶电泳。④用CaC12制备新鲜的大肠杆菌感受态细胞。⑤转化：将已转化的感受态细胞转移到琼脂培养基上。长出的克隆进行后续PCR鉴定。
2)质粒表达检测：①准备目的细胞。②目的细胞质粒转染：制成细胞悬液,接种于培养板中,根据Lipofectamine2000转染试剂使用说明书进行转染操作。转染后观察质粒上荧光标记基因的表达情况以判断感染效率。③蛋白印迹法检测目的基因表达,将收集的细胞提取蛋白进行蛋白印迹检测,同时提取7860细胞,和空质粒转染的293T细胞分别作为空白对照和阴性对照。
3)病毒包装和收集浓缩：细胞状态对于病毒包装至关重要,因此需要保证良好的细胞状态和较少的传代次数。收集浓缩后,进行病毒生物学滴度测定。
4)病毒滴度检测：使用荧光实时定量PCR法测定病毒滴度,通过比较control组和实验组的Ct值差异判断滴度值。
3. human-VEGF165-LV转染OECs、构建OECs-VEGF165基因工程细胞。
1)预实验：分四组进行,每组四个不同梯度的MOI (Multiply of Infection),共16孔,其中每种实验条件做三个复孔。每组均有不同梯度的MOI值。
2)免疫荧光检测：将未转染的OECs与转染后OECs传代接种,弃培养液,漂洗细胞两次,室温固定,PBS洗涤,加入正常山羊血清后进行孵育,洗涤,再在爬片上滴加Hoechst33342复染核,碱性甘油封固后荧光显微镜下观察。同时在染色过程中制作有阴性对照玻片,未加一抗。
3)Real time-PCR:①总RNA提取：取6孔板细胞,用吹打管吹至液体澄清,无细胞团块转至EP管中。用力摇晃试管、离心。用移液枪转上层水相于另EP管中,加等体积异丙醇,沉淀RNA,离心,用紫外光度检测RNA浓度。②RT-PCR:进行转染后OECs与未转染OECs细胞总RNA的逆转录。RT试剂盒中试剂解冻后,摇晃试管使其均匀,再稍离心,放于冰盒上操作。③Real-time PCR,融解曲线分析。
4)ELISA检测：在标准品孔及待测样本孔中分别加入酶联亲和物,包被微孔板覆膜、孵育后扣干孔内剩余残液,每孔依照次序分别加入底物,避光反应后每孔分别加入终止液,终止反应。使用酶标仪读取OD值。Logit-Log直线回归计算、绘制出标准曲线图。
研究结果：
1.嗅球OECs的取材、分离、培养、纯化与鉴定。
1)差速贴壁法所取出的原代细胞约在所有细胞中占98%,但是随着代数的增加,OECs的纯度很快下降,在第二代约占78%,到第三代纯度降至58%。
2)细胞形态和生长特性：纯化后OECs形态较为一致,主要呈梭性,约占细胞总数的90%以上；OECs也有其它形态,比较常见的有多突起型和不规则型。传代后OECs的形态发生改变,增殖形成的紧密连接消失,细胞也从梭性变成多突起形状。在培养液营养物质充分且完全的情况下,活性差的细胞渐渐凋亡,脱离瓶底,但对于大多数的细胞则可从多突起形态转化为增殖能力较强的梭形形态,继续增殖。
3)原代OECs的增殖能力强,但随着传代次数的增加,OECs纯度下降,活性也明显下降,并且细胞的形态变得多种多样,增殖变缓。
2.构建携带human-VEGF165基因的慢病毒。
1)质粒克隆载体的构建：从载有humanVEGF165基因的质粒上扩增出目的基因片段,并重新链接与用于慢病毒包装载体的质粒上,通过对连接上的阳性转化子测序,并与NCBI上的目的基因序列进行比对,得出包装质粒上连接的目的基因序列正确。
2)质粒表达目的基因检测：携带有EGFP蛋白基因做标记的质粒通过脂质体转染入293T细胞内,24小时后细胞内即可观察到明显的荧光,表明目的基因与EGFP表达正常。目的基因蛋白预测大小为22KD。通过Western Blot检测,从质粒转染成功的293T细胞提取的蛋白中可以检测到22KD附近处有条带,其大小和目的基因合成的蛋白分子量相吻合。这表明连接于克隆载体质粒上的目的基因在进入细胞后过表达成功。
3)合成病毒的滴度：通过Real-time PCR来检测病毒的滴度,得出在相当于病毒原液万分之一组(即梯度稀释中的10-4ul组)和Con组样品的Ct值出现2个循环左右差异,认为在10-4ul组样品中存在病毒颗粒,得出病毒滴度为2×108个／ml。
3. human-VEGF165-LV转染OECs、构建OECs-VEGF165基因工程细胞1)通过对不同浓度的病毒,以及不同转染条件的比较,得出对OECs慢病毒基因载体的最佳感染条件如下：对于合成的慢病毒,在MOI为10时即得到大于80%的感染效率；相对于普通培养基,Eni.s培养基并不能明显提高感染效率；Polybrene同样对病毒的感染效率改变不大。
2)转染后,OECs的生长活性受到影响。12小时内进入“休止期”,24小时后,细胞培养上清内死细胞增多,荧光不可见,48小时后,转染组的细胞死亡的更多,72小时后,少量细胞可见微弱荧光表达,96小时后,大部分细胞内出现EGFP的表达。
3)转染组MOI三个不同亚组,GFP阳性细胞数占总细胞数的比例大于80%。但是在转染后,10与20亚组细胞死亡较多,MOI为5亚组细胞死亡较少,细胞功能恢复快。对转染成功的OECs进行VEGFA免疫荧光染色,可观察外源VEGFA的表达。通过对转染效率,转染对OECs生长的影响,初步确定大量转染OECs时,MOI值为5是合适的选择。
结论
1.通过差速贴壁法纯化从嗅球提取出的OECs有高纯度和较好的活性,是体外研究OECs时提取和纯化原代细胞简便、经济的方法,适合进行相关的进一步研究。
2.随着原代细胞传代次数的增加,OECs的纯度和活性都明显下降,因而在OECs研究时,要选择合适传代次数下的OECs进行研究,使结果具有代表性。
3.携带有human-VEGF基因的慢病毒载体可以成功将目的基因转入OECs,并且由于慢病毒对原代细胞感染的高效性,在尽量小的影响OECs活性的情况下,达到较高的转染效率。
4.通过携带有human-VEGF基因的慢病毒载体感染OECs,构建出的human-VEGF-OECs基因工程细胞可稳定、高效表达VEGF165基因,合成的目的蛋白分泌到细胞外,符合VEGF165蛋白的生理特点,完成其在正常机体中的生物学作用。
Background
(?) High incidence and lagging treatment of leukoaraiosis
With the growing aging of population, the reinforcement of people's awareness about health and the development of imaging technology, leukoaraiosis (LA) has been attracting much attention. It is one of the most common cause of cognitive disorders of the elderly, in comparison with the incidence rate of1.7-8.6%of healthy elderly,78%-93%of the elderly patients with hypertension,30%-31%of the elderly patients with Alzheimer's disease and36%-100%of the elderly patients with stroke. The LADIS (Leukoaraiosis and Disability in the Elderly) study found that the body's ability level was related with the extent of white matter damage and the degree of white matter lesions was closely correlated with dementia disability assessment scores in the elderly not suffering from disability. Currently, most scholars believe that LA is due to the brain artery structural changes induced by age, hypertension and other vascular risk factors, which eventually leads to the brain ischemic injury. Hypertension and aging are the most important risk factors for LA.
LA is a chronic ischemic disease. After cerebral ischemia neurogenesis and angiogenesis are triggered as two major pathophysiological processes, which have an important role on promoting nerve function recovery after ischemic injuries. To improve the local blood supply is a fundamental solution to LA development, preventing the progression of the disease to the more serious (dementia, cerebral infarction).
(?) Research progress and analysis of vascular endothelial growth factor
Therapeutic angiogenesis is a new concept in recent years. It is the first to study on myocardial ischemia, aiming to stimulate the growth of the small blood vessels and to promote collateral circulation of the myocardium ischemic area, namely "self-bypass"."Currently, vascular endothelial growth factor (VEGF) is one of the most effective cytokines to stimulate the growth of blood vessels. The biological functions and characteristics of VEGF have attracted more and more attention. VEGF gene therapy is viewed as "molecular bypass surgery", which is expected to partially replace surgical bypass grafting and angioplasty. Study found that VEGF and its receptors upregulated after cerebral ischemia, intracerebral injection of VEGF led to the formation of new blood vessels in the brain and VEGFR-1expression in astrocytes and that VEGF also directly played a neuroprotective effect without relying angiogenesis, which contributed to the damage of cortical neurons from hypoxia and low sugar and stimulated the growth of axons.
There are rare reports about VEGF changes in the lesions and treatment of LA at home and abroad. The success of VEGF in the heart cerebrovascular experiments lay a solid foundation for the study of LA.It needs the further explorations about how to apply exogenous VEGF protein and VEGF gene therapy on cerebrovascular disease, improve the blood supply to the damaged brain tissue and reduce the extent of their ischemic, LA pathological changes mainly are ischemic demyelination and the decline in myelin content. Partial remyelination can control the progress of the LA and improve the clinical symptoms.
(?) Biological characteristics of olfactory ensheathing cells
Olfactory ensheathing cells (OECs) are cells of the dual characteristics of the astrocytes and Schwann cells and retain many of the characteristics of the developmental stages and plasticity. The olfactory system is the only renewable hub so far and its biological functions are extremely wide, exerting distinct facilitation on the growth, development and survival of neuron as well as promoting neurite outgrowth. Ramon-Cueto, studies have shown that rodents OECs can accelerate axonal regeneration and the remyelination of the demyelinated nerve fibers in rats with spinal cord injury. Therefore, OECs has opened up a new hope for cell transplantation in the treatment of CNS disorders.
Based on the biological role of OECs, many studies have focused on the application of olfactory ensheathing cell transplantation to treat demyelination of nerve tissue. Based on the study about the relationship between OECs and apoptosis, it can enhance the understanding of the therapeutic effect and mechanism of OECs and expand its range of treatment and improve their treatment.
(?) Composition and significance of the establishment of the genetically engineered cells OECs-VEGF
With the deepening of the study of the mechanism of the molecular level, gene therapy has become the new entry point for cerebrovascular disease prevention and treatment. Gene transfection technology enables the use of both treatment possible and the idea of composing genetically engineered cells is consequently established.
Based on the above analysis, it is envisaged that OECs are cultured. VEGF target gene is transferred to the recipient cell and builds OECs-VEGF gene engineering cells of rats with high expression of VEGF. The transplanted cells are injected into the brain. They not only can exert the synergic effect, but also resolve the low expression and poor continuity of VEGF which is the bottleneck problem to provide effective solution. LA cell transplantation and gene therapy to explore a new kind of practical breakthrough. Expected OECs are passaged and amplified in large quantities in the the LA model, and eventually differentiated to the nervous system, which has a role to promote axonal regeneration and repair of myelin. Meanwhile, after transfection the OECs have stronger biological activity and continually secret more VEGF to promote vascular endothelial regeneration, thereby improving the LA nerve tissue microcirculation, promoting angiogenesis, and reducing ischemic injury of the nerve tissue.
The purpose of this study was to separate, culture and purify OECs from the olfactory bulb. In addition, the lentivirus carrying human-VEGF165gene is construct and transfected OECs. Then OECs-VEGF gene engineered cells are established.
Objectives
1. To explore the method of introducing human-VEGF165gene into OECs and analysis the feasibility of building the OECs-VEGF genetically engineered cells.
2. To research the technique of purifying OECs from the olfactory bulb isolated rat.
3. To construct the lentivirus vector carrying the human-VEGF165gene
4. To building the OECs-VEGF165genetically engineered cells by human-VEGF165-LV transfecting OECs
Methods
1. Sampling, separation, cultivation, purification and identification of OECs from olfactory bulb
Male SD rats were decapitated. The olfactory nerve layer and granular layer of the olfactory bulb were taken, cut into pieces and digested in trypsin in the incubator. DF12containing FBS terminated the digestion. It was blown and beaten. Draw the upper suspension to be inoculated after standing a while and repeat three times. The OECs were mainly purified by differential adherence method. Cell suspension was joined with dual antibody and cultivated for2d so ld.The cultivated cells were adjusted, planted and adhered. Medium was changed every3days. Take the primary cells, prepare a cell suspension which was inoculated in24-well plates, count the total number of cells, average for the following nine days and make the growth curve.
The purity of primary OECs is identified by immunofluorescence. Remove the culture medium, rinse repeatedly, fix and incubate twice. The nuclear was stained with Hoechst33342. Alkaline glycerol was dropped on glass slides. After p75staining, sealed and observed with fluorescence microscopy. Counted the number of nuclear and positive cells in high magnification view and calculated the ratio of the positive cells, which is the purity of OECs.
2. Construct the lentivirus carrying the human-VEGF165gene
A lentiviral vector carrying the desired gene was constructed by three plasmid co-transfecting of293T cells with the lentivirus packaging kit.
1) Cloning Construction:①The carrier preparation which overexpressed objective gene:catching the target gene by PCR, digesting the vector and exchange and transforming its product into competent cells of bacteria.②Making the information about target gene and vector clear.③DNA agarose gel electrophoresis.④Preparing fresh E. coli competent cells by using the CaCl2.⑤Transferring the transformed competent cells to the agar medium. Grown clones were identified by subsequent PCR.
2) Plasmid expression detection:①Preparation of the purpose cells.②Target cell plasmid transfection:preparing a cell suspension, seeding in culture plates and transfecting according to the Lipofectamine2000transfection reagent manual operation. In order to determine the efficiency of infection was judged by fluorescently labeled plasmid gene expression.③Proteins were extracted from the collected cells and were detected by western blotting, Meanwhile,7860cells and empty plasmid-transfected293T cells were used as blank control and the negative control.
3) Viral packaging, collecting and concentrating:the state of cells was essential for viral packaging, so we needed ensure a good state of cells and less number of passages. After collecting and concentrating, viral biological titer was determinated.
4) Viral titer detection:the titer was detected by fluorescence real-time quantitative PCR and determined by comparing the Ct value differences between the control group and the test group.
3. Building the OECs-VEGF165genetically engineered cells by human-VEGF165-LV transfecting OECs
1) Pre-experiment:four groups, each of which had four different gradients MOI (Multiply of Infection), a total of16holes, there were three wells for every experimental condition. MOI value in each group had a different gradient.
2) Immunofluorescence detection:After inoculation and passage untransfected and transfected OECs, discard the culture medium, rinse the cells twice, fix twice at room temperature, wash3times with PBS and incubate after addition of normal goat serum. Then wash, stain nucleus in climbing slices with Hoechst33342, seal with alkaline glycerol and observe under fluorescence microscopy. At the same time, the slides without adding antibody in the dyeing process were used as the negative control slides.
3) Real time-PCR:①Extraction of total RNA:blow the cells in6-well plates until they were clear then transfer them to EP tubes. Firmly shake and centrifuge the test tubes. By pipette transfer upper aqueous phase to another EP tube, add an equal volume of isopropanol to precipitate the RNA, centrifuge, and detectRNA concentration with ultraviolet degrees.②RT-PCR:Total RNA of the untransfected and transfected OECs was transcribed reversely after transfection. After thawing the reagents of RT kit, shake up the test tube, slightly centrifugate and perform on ice.③Real-time PCR. Melting curve analysis.
4) ELISA detection:enzyme-linked pro and material were added to the standard holes and test sample hole. After microplate coated and drained after incubation, the substrate was added to each well in accordance with the order respectively. After kept in the dark, the liquid to terminate the reaction was added. OD value was shown with a microplate reader. Calculated with Logit-Log linear regression and draw a standard graph.
Results
1. Sampling, separation, cultivation, purification and identification of OECs from olfactory bulb
1) The primary cells cultivated by differential adherent method approximately accounted for98%of all cells. More the times of passage were, lower the purity of OECs was. In the second generation the purity of OECs declined to78%and the third was58%.
2) Cellular morphology and growth characteristics:the cellular morphology after purification was consistent, mainly spindle-shaped, accounting for about90%of the total cells. There were other common forms, multiple swelling and irregular. Cellular morphology changed, tight junctions disappeared and cells changed from spindle-shaped to multiple swelling after passage. In the case of culture liquid nutrients fully and completely, cells with poor activity died gradually and seperated from the bottom. But the most of the cells could be transformed from multiple projections to spindle-shaped form with stronger proliferation ability and continued to proliferate.
3) The proliferative capacity of primary OECs was strong. But with the times of passages increased, the purity of OECs decreased. The cell shape diversified and the proliferation slowed.
2. To construct the lentivirus carrying the human-VEGF165gene
1) Construction of plasmid cloning vector. The target gene fragment was amplified from the plasmid containing humanVEGF165gene, and re-linked to the plasmid used as lentivirus packaging vectors. By sequencing the positive transformants and comparing with the NCBI on the target gene sequences, the target gene sequence was clear.
2) Detection of plasmid expressing the gene. The plasmids labeled with the EGFP protein gene were transfected into293T cells by liposome. Significant fluorescence the cells could be observed after24hours, which indicated that the expression of target gene and EGFP was normal. The predicted size of the target gene protein was22KD. By Western Blot analysis, the protein extracted from plasmid transfected293T cells could be detected22kD near bands, which was consistent with the protein synthesized by target. This indicates that the connection was successful.
3) Synthesis of viral titer. Ct value appeared2circulating difference between one ten thousandth group (i.e. gradient group the10-4ul dilution) and Con group by real-time PCR. It was considered that there were virus particles in the samples10-4ul and virus titer of2×108/ml was obtained.
3. Building the OECs-VEGF165genetically engineered cells by human-VEGF165-LV transfecting OECs
1) From different concentrations of the virus and the comparison of different transfection conditions, the best infection conditions of lentiviral gene vector of OECs were as follows:for lentiviral synthetic, at MOI=10to obtain greater than80%of the efficiency of infectionat; relatived to the normal medium, Eni.s medium did not significantly improve the infection efficiency; Polybrene also little changed in the efficiency of virus infection.
2) After transfection, the growth activity of OECs was affected."Telogen" within12hours, after24hours, an increase in the dead cells in the cell culture supernatant which was not visible under fluorescence, and48hours more died cells in transfection group. After72hours the weak fluorescence was visible in a small amount of cells. After96hours, the majority of cells expressed EGFP.
3) GFP-positive cells accounted for more than80%in the three transfection sub-groups with different MOI. But after transfection,10and20subgroups cells died more. Cells of MOI5died less and cell function recovered quickly. VEGFA immunofluorescence staining was performed in the transfected OECs and the expression of the exogenous VEGFA was observed. From transfection efficiency and the impact of transfection on the growth of OECs, MOI=5was a more appropriate choice.
Conclusion
1. OECs from the olfactory bulb which are purified by differential adherent method appear high purity and good activity. That is a simple and economical way for extraction and purification of primary cells in vitro studies, which is suitable for further relevant studies.
2. With the times of passages increase, the purity of OECs declines significantly. Thus to make the results more representative, it is important to select the appropriate number of passages during OECs research.
3. Target gene can be transferred into OECs by lentiviral vector carrying the human-VEGF gene. Because of the high infection efficacy of lentivirus in primary cells, higher transfection efficiency can be achieved in the case of the influence of the activity of OECs as small as possible.
4. OECs are infected by the lentiviral vector carrying the human-VEGF gene to construct the human-VEGF-OECs genetically engineered cells, which can express of VEGF165gene stably and efficiently. The synthetic protein can be secreted to the extracellular which has the same physiological characteristic as VEGF165protein and can play a biological role in a normal body.
引文
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84刘晓阳,孙建民,崔新刚,蒋振松,王国栋.嗅鞘细胞联合VEGF对大鼠损伤脊髓的保护作用.中国修复重建外科杂志,2011,25(6)：699-704.
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