核素报告基因显像监测转基因骨髓间充干细胞移植治疗的基础研究
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摘要
目的
构建携带HSV1-TK和BDNF的重组真核质粒表达载体pcDNA3.1-TK-IRES-BDNF和重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP,分别将其转染体外培养的大鼠BMSCs,探讨不同载体对BMSCs的转染效率、转染细胞的增殖能力和向神经元样细胞诱导分化能力、两目的基因的表达相关性,以及转染报告基因(HSV1-TK)的BMSCs对131I-FIAU的体外摄取。为下一步选择合适的载体活体监测转基因BMSCs治疗脑梗死提供实验基础。
方法
1.不同重组载体的构建和SD大鼠BMSCs的培养及鉴定
1.1.重组真核质粒表达载体pcDNA3.1-TK-IRES-BDNF的构建及鉴定
根据Gene bank上的CDs区,设计并合成HSV1-TK、IRES、BDNF的全长扩增引物,并在引物两端加入与真核质粒表达载体pcDNA3.1+的多克隆位点(multiple cloning sites,MCS)相对应的酶切位点。应用聚合酶联反应(polymerase chain reaction,PCR)技术分别从重组质粒pcDNA3.1-TK、pIRES-EGFP中扩增获得TK和IRES的cDNA序列；用RT-PCR技术从1周龄SD大鼠脑组织中提取、扩增BDNF的cDNA序列。对上述目的基因全长片段进行双酶切后,依次插入到经过相对应酶切的质粒载体pcDNA3.1+的MSC中,每一步得到的重组质粒均经过酶切鉴定,最后进行序列分析。
1.2.重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP的构建及鉴定
以含巨细胞病毒(cytomegalovirus, CMV)启动子的表达质粒pDC316-EGFP为载体,将偶联基因TK-IRES-BDNF插入MCS,构建重组腺病毒表达质粒载体pDC316-TK-IRES-BDNF-EGFP。以pDC316-TK-IRES-BDNF-EGFP为重组表达质粒,与重组腺病毒(Adenovirus, Ad)骨架质粒共转染293T细胞,包装生成腺病毒重组体Ad5-TK-IRES-BDNF-EGFP,以Ad5-EGFP为对照病毒。本研究部分与上海鸣宏生物有限公司合作完成。
1.3.SD大鼠BMSCs的培养及鉴定
分离4-6周健康SD大鼠股骨和胫骨,用含15%胎牛血清的DMEM-F12培养基冲出骨髓液,密度梯度离心后,接种于塑料培养瓶中,培养出的BMSCs连续传3-4代,细胞纯化后,倒置显微镜下观察细胞形态,并通过免疫细胞化学检测表面抗原CD44和CD34的表达。
2.不同载体转染BMSCs的实验研究
2.1.不同载体对BMSCs转染效率的测定
在相同转染条件下,用脂质体介导重组真核质粒表达载体pcDNA3.1-EGFP转染BMSCs,在绿色荧光显微镜下观察增强绿色荧光蛋白(enhanced green fluorenscent protein, EGFP)的表达时间和表达阳性率,间接反映脂质体介导重组质粒载体pcDNA3.1-TK-IRES-BDNF对BMSCs的转染效率和表达时间。重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP在不同转染复数(multiplicity of infection, MOI)下转染BMSCs,荧光显微镜下观察EGFP的表达时间和表达阳性率。
2.2.不同载体转染BMSCs后细胞增殖能力、向神经元样细胞分化能力的检测
不同重组基因载体转染BMSCs后,在不同时间内,倒置显微镜下观察细胞形态和密度变化,MTT比色法检测转染细胞的增殖能力。对转染重组腺病毒的BMSCs加入碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)和表皮生长因子(epidermal growth factor, EGF),不同时间下在倒置显微镜下观察细胞形态变化,检测转染细胞向神经元样细胞的分化能力。
2.3.不同载体转染BMSCs后目的基因表达的检测
重组质粒载体pcDNA3.1-TK-IRES-BDNF转染BMSCs后用RT-PCR检测TK基因的转录,Western blot检测BDNF蛋白的表达；不同MOI的重组腺病毒Ad5-TK-IRES-BDNF-EGFP转染BMSCs后,实时定量聚合酶联反应(real-time quantitativepolymerase chain reaction, RQ-PCR)和2-△△CT方法分析两目的基因HSV1-TK与BDNF的相对表达量(CT),并进行相关性分析；Western blot检测并分析HSV1-TK与BDNF的蛋白水平表达。
3.131I-FIAU的标记及BMSCs转染不同载体后对131I-FIAU的摄取
3.1.131I-FIAU的标记及质量鉴定
利用Iodogen固相氧化法对FAU进行放射性碘标记,标记产物用Sep-Pak-C-18反相层析色谱小柱进行纯化及标记率分析,TLC-SG硅胶板薄层层析法测定标记产物的放射化学纯度,并观察131I-FIAU在37℃正常人新鲜血清中放置4h、12h和24h后的体外稳定性。
3.2.不同载体转染BMSCs后对mI-FIAU的摄取研究
不同载体转染BMSCs 48h后,加入纯化后的131I-FIAU,在不同时间分别计数细胞培养基上清液中的放射性计数和消化后细胞的放射性计数,细胞摄取结果为：摄取率=C细胞悬液/(C细胞悬液+C细胞培养基)×100%。
第一组实验,重组真核质粒表达载体pcDNA3.1-TK-IRES-BDNF转染BMSCs 48h后,加入纯化后的131I-FIAU。在一定观察时间内,分析转染重组质粒的BMSCs对131I-FIAU的摄取数值随时间的变化,以转染质粒pcDNA3.1+为对照组。
第二组实验,在MOI分别为O、50、100、150、200、250转染BMSCs 48h后,加入纯化后的131I-FIAU。在相同时间下分析不同MOI的重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP转染BMSCs对131I-FIAU的摄取研究。
第三组实验,结合上述实验不同MOI的重组腺病毒Ad5-TK-IRES-BDNF-EGFP对BMSCs的转染效率,MTT对增殖能力的分析结果,不同MOI的重组腺病毒在相同时间下对131I-FIAU的摄取结果。以最佳MOI的重组腺病毒Ad5-TK-IRES-BDNF-EGFP转染BMSCs,分析转染重组腺病毒的BMSCs对131I-FIAU的摄取数值随时间变化的关系,以转染无效病毒Ad5-EGFP为对照组。
4.统计学方法
数据分析采用SPSS11.0软件进行,线性相关采用Pearson分析,两样本均数比较采用双侧t检验,以P＜0.05为有统计学意义。
结果
1.不同重组载体的构建和SD大鼠BMSCs的培养及鉴定
1.1.重组真核质粒表达载体pcDNA3.1-TK-IRES-BDNF的构建及鉴定
重组真核质粒表达载体pcDNA3.1-TK-IRES-BDNF经PCR、酶切和序列分析鉴定与预期设计一致。
1.2.重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP的构建及鉴定
由上海鸣鸿生物有限公司协作构建的重组表达质粒pDC316-TK-IRES-BDNF-EGFP经PCR、酶切鉴定和测序鉴定与预期设计一致；包装生成的重组腺病毒Ad5-TK-IRES-BDNF-EGFP的活病毒数量达到2.0×1010 PFU (plaque formation unit)/ml,对照重组腺病毒Ad5-EGFP的活病毒数量为3.6×1010PFU/ml。
1.3.大鼠BMSCs的培养及鉴定
采用密度梯度离心法获得到的BMSCs传到3-4代后,可获得较高纯度和保持良好的增殖能力；细胞免疫化学鉴定,CD44在95%以上的BMSCs表面表达,而CD34则未见明显表达。
2.不同载体转染体外培养的BMSCs实验研究
2.1.不同载体对BMSCs转染效率的测定
以阳离子脂质体(μl)与重组质粒载体(μg) pcDNA3.1-EGFP在2.5：1时转染BMSCs,48h时EGFP有较高表达效率,在随后的观察时间内EGFP的表达开始下降；不同MOI的重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP转染BMSCs后,绿色荧光显微镜下观察,在24h开始表达EGFP,48h达到高峰,120h开始下降；MOI为150时在转染效率可大于90%。
2.2.不同载体转染BMSC后细胞增殖能力、向神经元样细胞分化能力的检测
不同载体转染BMSCs后,在一定时间内于倒置显微镜下观察。发现重组质粒载体pcDNA3.1-TK-IRES-BDNF转染BMSCs后细胞密度明显减低,有较多细胞漂浮,台盘兰染色证实为死亡细胞。而重组腺病毒Ad5-TK-IRES-BDNF-EGFP的MOI为250时转染BMSCs后,在观察时间内细胞形态和密度均无明显变化,细胞培养基中仅可见极个别漂浮细胞。MTT比色法亦证实重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP对BMSCs的毒性作用较小, MOI在150时细胞存活率仍大于98%。重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP转染BMSCs的同时在细胞培养基中加入bFGF和EGF,倒置显微镜下观察细胞形态变化,发现转染重组腺病毒组和未转染重组腺病毒组的BMSCs均保持向神经元样细胞的良好分化能力,两组相比无明显差别。
2.3.不同载体转染BMSCs目的基因表达的检测
重组真核质粒表达载体pcDNA3.1-TK-IRES-BDNF转染BMSCs 48h后,通过RT-PCR技术可检测到HSV1-TK的表达,Western blot检测到BDNF的表达。不同MOI的重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP转染BMSCs后,通过RQ-PCR检测到两目的基因的表达,并且随着病毒MOI的增加表达增强。HSV1-TK和BNDF两目的基因的表达有良好的线性相关(r=0.973, p<0.05, n=3)。Western blot亦检测到了HSV1-TK和BNDF两目的基因的表达,随着病毒MOI的增加,基因表达也在增加。
3.131I-FIAU的标记及不同载体转染BMSCs后对131I-FIAU的摄取研究
3.1.131I-FIAU的标记及质量鉴定
Iodogen固相氧化法能有效标记FAU；经测定,标记物的标记率为(64.35±4.89)%(n=5)。TLC-SG硅胶板层析法测定,131I-FIAU的放射化学纯度为(98.62±0.62)%(n=5).131I-FIAU在正常人血清中37℃中孵育24h后,放射化学纯度仍大于95%。
3.2.不同载体转染BMSCs后对131I-FIAU的细胞摄取研究
第一组实验,重组质粒载体pcDNA3.1-TK-IRES-BDNF转染BMSCs后对131I-FIAU的摄取研究,以转染质粒pcDNA3.1+为对照组。
重组质粒载体pcDNA3.1-TK-IRES-BDNF转染BMSCs 48h后,加入纯化后的131I-FIAU,分析5h内对131I-FIAU的摄取。研究发现在前3h随着时间的延长摄取增加,在3h对131I-FIAU的摄取达到平台期,值为(4.15±0.074)%,n=3,在随后的观察时间内摄取数值增加不明显。在各个观察时间点转染目的质粒组与对照质粒组对131I-FIAU的摄取数值相比,有统计学意义(t=9.29-232.75,p<0.05,n=3)。
第二组实验,MOI分别为0、50、100、150、200、250的重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP转染BMSCs后,分析相同时间对131I-FIAU的细胞摄取。
不同MOI的重组腺病毒Ad5-TK-IRES-BDNF-EGFP转染BMSCs 48h后,在同一时间点3h时分析转染细胞对131I-FIAU的摄取。研究发现重组腺病毒随着MOI的增加对131I-FIAU的摄取增加,在MOI为150时达到平台期,随后随着MOI的增加转染重组腺病毒的BMSCs对131I-FIAU摄取增加不明显。
第三组实验中,相同MOI重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP转染BMSCs后,分析转染BMSCs不同时间对131I-FIAU的摄研究,以转染对照病毒Ad5-EGFP为对照组。
结合上述转染效率、MTT等实验结果,确定重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP的MOI为150时是对BMSCs最佳转染效率。MOI为150转染BMSCs 48h后,加入纯化后的131I-FIAU,分析不同时间转染重组腺病毒的BMSCs对131I-FIAU的摄取研究。结果发现在前3h内,转染了重组腺病毒Ad5-TK-IRES-BDNF-EGFP的BMSCs随着时间的延长,其摄取131I-FIAU也在增加,在3h摄取数值可达(31.42±0.46)%(n=3),此后随着时间的延长,摄取数值增加不明显。在观察时间内各时间点转染重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP的BMSCs对131I-FIAU的摄取均显著高于对照组,两组相比具有统计学意义(t=23.06-173.83,P<0.05,n=3)。
结论
本研究通过体外比较携带报告基因HSV1-TK和治疗基因BDNF的不同载体转染BMSCs实验研究,证实重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP是一种可以携带较大容量碱基,对BMSCs有较高转染效率,转染重组腺病毒后的BMSCs仍保持良好的增殖能力和向神经元样细胞诱导分化能力,IRES介导上游报告基因的表达可间接反映下游治疗基因的表达,二者具有良好的线性相关。利用Indogen固相氧化法对FAU进行放射性核素131I标记,标记产物用Sep-Pak-C-18反相层析色谱小柱进行纯化,是一种简单的标记及纯化技术,可得到较高的放射化学纯度。重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP转染BMSCs可表达有活性的HSV1-TK,能有效介导BMSCs摄取131I-FIAU,在一定的MOI范围内其摄取数值随着MOI的增加而增加,有明显的剂量依赖性；此外,在一定的观察时间内随着时间的延长,对131I-FIAU的摄取数值亦在增加,显著高于对照组。本研究为后续以重组腺病毒Ad5-TK-IRES-BDNF-EGFP为载体介导的放射性核素报告基因活体监测踪转基因BMSCs治疗脑梗死的干细胞有效归巢数量、分化和功能表达过程,以及对外源基因的表达部位和时间的活体评估奠定了良好的实验基础。
Purpose
Constructing recombinant eukaryotic plasmid expression vector pcDNA3.1-TK-IRES-BDNF and recombinant adenovirus vector Ad5-TK-IRES-BDNF-EGFP, to assess the multiplication capacity, differentiation potential, uptake of 131I-FIAU capacity of rat BMSCs transfected with recombinant vectors, finally selecting a appropriate vector for further monitoring the genetically modified BMSCs in vivo
Methods
1. Construct different vectors and culture rat BMSCs
1.1. Construct and identify vector pcDNA3.1-TK-IRES-BDNF
The primers of HSV1-TK, IRES and BDNF were designed and sythesised according to Gene bank. Restriction enzyme sites were added into the both ends, which were matched with the multiple cloning sites (MCS) of eukaryotic expression plasmid vector pcDNA3.1+. The cDNA of target genes HSV1-TK and IRES were obtained from recombinant plasmid pcDNA3.1-TK, pIRES-EGFP by polymerase chain reaction (PCR) separately. The cDNA of target gene BDNF was obtained from a week-old SD rat brain tissue by RT-PCR technology. Three target genes were restricted with respective enzymes, and subcloned into the eukaryote plasmid pcDNA3.1+restricted with the same enzymes. Restriction analysis and sequencing were used to confirm the recombinant plasmid vector pcDNA3.1-TK-IRES-BDNF.
1.2. Construct and identify vector Ad5-TK-IRES-BDNF-EGFP
The plasmid vector, pDC316-TK-IRES-BDNF-EGFP, and the virus vector, E1/E3-deleted replication-defective recombinant adenovirus type 5 (Ad5-TK-IRES-BDNF-EGFP), carrying the HSV1-TK-IRES-BDNF coupling gene under the transcriptional control of the cytomegalovirus (CMV) promoter, were constructed and purified in the Vector Gene Technology Company Ltd. in Shanghai.
1.3. Culture rat BMSCs
The femur and tibia were isolated from health SD rat aged 4-6 weeks, the bone marrow were washed out with DMEM-F12 containg 15%FBS, and operated into the cultured bottles after density gradient centrifugation. BMSCs were cultivated and expanded in vitro. The cell morphology were observed under inverted microscope at different times; expression of surface antigens CD44 and CD34 were detected by immunocytochemistry.
2. BMSCs were transfected with different recombinant vectors in vitro
2.1. The transfection efficiency for BMSCs with different vectors
BMSCs were transfected with recombinant plasmid vector pcDNA3.1-EGFP, enhanced green fluorenscent protein (EGFP) expression were observed under fluorescence microscope, transfection efficiency was analyzed with flow cytometry (FCM). BMSCs were transfected with recombinant vector Ad5-TK-IRES-BDNF-EGFP at different multiplicity of infection (MOI), from 0,50,100,150,200 and 250, transfection efficiency was assayed by fluorescence microscope.
2.2. The proliferation and differentiation of genetically moditified BMSCs with different vectors
After BMSCs were transfected with different vectors, morphology and density changes of genetically moditified BMSCs were observed under inverted microscope at different times. MTT were used to detect the proliferation, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were added into the genetically moditified BMSCs, the morphology and the potential of differentiation were observed under inverted microscope.
2.3. The expression of target genes
When BMSCs were transfected with plasmid vector pCDNA3.1-TK-IRES-BDNF, RT-PCR and Western blot analysis were used to detect the expression of target genes in BMSCs. Real-time quantitative polymerase chain reaction (RQ-PCR) and Western blot were used to detect the expression of target gene in BMSCs transfected with different MOI of recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP.
3. The radioiodination of FAU and uptake 131I-FIAU for BMSCs transfected with different vectors
3.1. The radioiodination of FAU
Using solid phase oxidation with Iodogen, FAU was labeled with 131I. The product, 131I-FIAU, was purified on a reverse-phase Sep-Pak C-18 column and the labeling efficiency was then assayed. Moreover, the peak fractions were identified for the radiochemical purity assessment. To study the stability in vitro, the 131I-FIAU was incubated in serum at 37℃for 4,12 and 24 hours before the aliquots were removed for radiochemical purity analysis.
3.2. Uptake of 131I-FIAU for transgenic BMSCs by gamma counter
BMSCs were transfected with different vectors,48h later, the purified 131I-FIAU were added into the genetically modified BMSCs. The radiocounting of cell culture medium and digested cell were counted by gamma counter separately, uptake rate= C digested cell/(C digested cell+C cell culture medium)×100%.
PartⅠ, BMSCs were transfected with recombinant eukaryotic plasmid vector pcDNA3.1-TK-IRES-BDNF,48h later, adding purified 131I-FIAU. Uptake of 131I-FIAU was observed in a certain period of time, the control group was pcDNA3.1-EGFP.
PartⅡ, BMSCs were transfected with recombinant adenovirus vector Ad5-TK-IRES-BDNF-EGFP, the MOI was 0,50,100,150,200 and 250,48h later, adding the purified 131I-FIAU. Uptake of 131I-FIAU was observed at the same time, control group was Ad5-EGFP.
PartⅢ, the best MOI was obtained from the above-mentioned experiments, and then BMSCs were transfected with recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP in the best MOI. Uptake of 131I-FIAU was observed in a certain period of time, the control group was Ad5-EGFP.
4. Statistical methods
Data were pcocessed by SPSS 11.0 software.
Results
1. Construct different vectors and culture rat BMSCs
1.1. Construct and identify plasmid vector pcDNA3.1-TK-IRES-BDNF
The identification of recombinant eukaryotic expression plasmid vector pcDNA3.1-TK-IRES-BDNF by PCR, enzyme digestion and sequence analysis, and the result was matching with expectations.
1.2. Construct and identify vector Ad5-TK-IRES-BDNF-EGFP
The recombinant plasmid, pcDNA3.1-TK-IRES-BDNF, were identified by PCR、restriction enzyme digestion and sequencing. The titer of packaged recombinant adenovirus, Ad5-TK-IRES-BDNF-EGFP, was 2.0×1010 PFU/ml; A replication-defective adenovirus type 5 (Ad5-EGFP) was used as a control with the titer of 3.6×1010PFU/ml.
1.3. Culture and identify rat BMSCs
BMSCs can obtain high purity and good proliferation by gradient centrifugation. The surface antigen CD44 were observed in more than 95% of the BMSCs, while CD34 were no observed by immunohistochemistry.
2. BMSCs were transfected with different recombinant vectors in vitro
2.1. The transfection efficiency for BMSCs with different vectors
There was high transfection efficiency for BMSCs transfected with recombinant plasmid pcDNA3.1-EGFP, when the ratio between liposome (μl) and the recombinant plasmid (μg) was 2.5:1. When the MOI was 150, transfection efficiency for BMSCs transfected with recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP can reach peak, at the 48h, the transfection efficiency was more than 90%.
2.2. The proliferation and differentiation of transgenic BMSCs with different vectors
BMSCs transfected with recombinant plasmid pcDNA3.1-TK-IRES-BDNF, the cell density were increased seriously and there have many floating cells, death cells were confirmed by placenta blue staining. BMSCs transfected with recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP, the MOI was 0,50,100,150,200 and 250 separately. Little floating cell were observed under inverted microscope, when the MOI was 250. There have little toxicity for BMSC transected with recombinant adenovirus by MTT assay. The cell survival rate was more than 98% when the MOI was 150.
After BMSCs were transfected with recombinant adenovirus vector Ad5-TK-IRES-BDNF-EGFP, non-transfected recombinant adenovirus group was control, adding bFGF and EGF separately. The morphology change of genetically moditified BMSCs were observed under inverted microscope in a certain period of time, and the difference was no founded between adenovirus transfection group and non-transfected recombinant adenovirus group. Transgenic BMSCs remain fine ability of differentiation into neuron-like cells.
2.3. The expression of target gene were detected
The expression of TK and BDNF were detected with RT-PCR and Western blot separately, when BMSCs were transfected with recombinant eukaryotic expression vector pcDNA3.1-TK-IRES-BDNF,48h later. BMSCs were transfected with different MOI, the mRNA of TK and BDNF were deteted by RQ-PCR, expression of target gene upgrade with the increased MOI for a range. There was a strong relation between the mRNA expression of TK and BDNF for transfected BMSCs(r=0.973,p<0.05, n=3). The expression of target gene was detected by Western blot also.
3. The radioiodination of FAU and uptake 131I-FIAU for BMSCs transfected with different vectors
3.1. Radiolabeling and stability
FAU could be labeled efficiently by solid phase oxidation with Iodogen. The radiolabeling efficiency of 131I-FIAU was (64.35±4.89)%(n=5). After purification on Sep-Pak C-18 column, the radiochemical purity of the final product 131I-FIAU was (98.62±0.62)%(n=5). The radiochemical purity of 131I-FIAU remained above 95% after incubated in serum at 37℃for 4,12 and 24 hours, which indicated 131I-FIAU was stable in serum.
3.2. Uptake of 131I-FIAU for genetically modified BMSCs by gamma counter
PartⅠ, BMSCs were transfected with recombinant eukaryotic plasmid vector pcDNA3.1-TK-IRES-BDNF,48h later, adding purified 131I-FIAU. Uptake of 131I-FIAU was observed in 5 hours. Uptake of 131I-FIAU for genetically modified BMSCs was increased with time extension in 3 hours, there has significant increase compared with the control.
PartⅡ, BMSCs were transfected with recombinant adenovirus vector Ad5-TK-IRES-BDNF-EGFP, the MOI was 0,50,100,150,200 and 250 separately,48h later, adding the purified 131I-FIAU. Uptake of 131I-FIAU was observed in 3 hours. Uptake of 131I-FIAU for genetically modified BMSCs was increased with the MOI raising, uptake was reach peak at 3h point, when the MOI was 150.
PartⅢ, the best MOI was obtained from the above-mentioned experiment, the best MOI was 150. Then BMSCs were transfected with recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP at MOI was 150. Uptake of 131I-FIAU was observed in 5 hours, uptake of 131I-FIAU for genetically moditified BMSCs was increased with time extension in 3 hours. There has significance between genetically modified BMSCs and control BMSCs for uptake of131I-FIAU at all the time points(t=20.74-98.16, p<0.05, n=3).
Conclusion
Recombinant adenovirus can contain more bases, get higher transfection efficiency, lower toxic for BMSCs, and remain fine differentiation into neuron-like cells. There has a strong relation between the mRNA expression of HSV1-TK and BDNF in genetically modified BMSCs. The expressions of upstream reporter gene can indirect response to the downstream therapeutic gene by IRES. FAU could be labeled efficiently by solid phase oxidation with Iodogen; the high radiochemical purity for product can obtain by Sep-Pak-C-18 reversed-phase chromatography. BMSCs transfected with recombinant adenovirus vector Ad5-TK-IRES-BDNF-EGFP can express HSV1-TK possessed fine activity, effective uptake of 131I-FIAU, there has significance between genetically modified BMSCs and control BMSCs for uptake of 131I-FIAU at all the time points. This study indicates that recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP may be a suitable gene vector for research tracing genetically modified stem cells.
构建携带HSV1-TK和BDNF的重组真核质粒表达载体pcDNA3.1-TK-IRES-BDNF和重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP,分别将其转染体外培养的大鼠BMSCs,探讨不同载体对BMSCs的转染效率、转染细胞的增殖能力和向神经元样细胞诱导分化能力、两目的基因的表达相关性,以及转染报告基因(HSV1-TK)的BMSCs对131I-FIAU的体外摄取。为下一步选择合适的载体活体监测转基因BMSCs治疗脑梗死提供实验基础。
方法
1.不同重组载体的构建和SD大鼠BMSCs的培养及鉴定
1.1.重组真核质粒表达载体pcDNA3.1-TK-IRES-BDNF的构建及鉴定
根据Gene bank上的CDs区,设计并合成HSV1-TK、IRES、BDNF的全长扩增引物,并在引物两端加入与真核质粒表达载体pcDNA3.1+的多克隆位点(multiple cloning sites,MCS)相对应的酶切位点。应用聚合酶联反应(polymerase chain reaction,PCR)技术分别从重组质粒pcDNA3.1-TK、pIRES-EGFP中扩增获得TK和IRES的cDNA序列；用RT-PCR技术从1周龄SD大鼠脑组织中提取、扩增BDNF的cDNA序列。对上述目的基因全长片段进行双酶切后,依次插入到经过相对应酶切的质粒载体pcDNA3.1+的MSC中,每一步得到的重组质粒均经过酶切鉴定,最后进行序列分析。
1.2.重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP的构建及鉴定
以含巨细胞病毒(cytomegalovirus, CMV)启动子的表达质粒pDC316-EGFP为载体,将偶联基因TK-IRES-BDNF插入MCS,构建重组腺病毒表达质粒载体pDC316-TK-IRES-BDNF-EGFP。以pDC316-TK-IRES-BDNF-EGFP为重组表达质粒,与重组腺病毒(Adenovirus, Ad)骨架质粒共转染293T细胞,包装生成腺病毒重组体Ad5-TK-IRES-BDNF-EGFP,以Ad5-EGFP为对照病毒。本研究部分与上海鸣宏生物有限公司合作完成。
1.3.SD大鼠BMSCs的培养及鉴定
分离4-6周健康SD大鼠股骨和胫骨,用含15%胎牛血清的DMEM-F12培养基冲出骨髓液,密度梯度离心后,接种于塑料培养瓶中,培养出的BMSCs连续传3-4代,细胞纯化后,倒置显微镜下观察细胞形态,并通过免疫细胞化学检测表面抗原CD44和CD34的表达。
2.不同载体转染BMSCs的实验研究
2.1.不同载体对BMSCs转染效率的测定
在相同转染条件下,用脂质体介导重组真核质粒表达载体pcDNA3.1-EGFP转染BMSCs,在绿色荧光显微镜下观察增强绿色荧光蛋白(enhanced green fluorenscent protein, EGFP)的表达时间和表达阳性率,间接反映脂质体介导重组质粒载体pcDNA3.1-TK-IRES-BDNF对BMSCs的转染效率和表达时间。重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP在不同转染复数(multiplicity of infection, MOI)下转染BMSCs,荧光显微镜下观察EGFP的表达时间和表达阳性率。
2.2.不同载体转染BMSCs后细胞增殖能力、向神经元样细胞分化能力的检测
不同重组基因载体转染BMSCs后,在不同时间内,倒置显微镜下观察细胞形态和密度变化,MTT比色法检测转染细胞的增殖能力。对转染重组腺病毒的BMSCs加入碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)和表皮生长因子(epidermal growth factor, EGF),不同时间下在倒置显微镜下观察细胞形态变化,检测转染细胞向神经元样细胞的分化能力。
2.3.不同载体转染BMSCs后目的基因表达的检测
重组质粒载体pcDNA3.1-TK-IRES-BDNF转染BMSCs后用RT-PCR检测TK基因的转录,Western blot检测BDNF蛋白的表达；不同MOI的重组腺病毒Ad5-TK-IRES-BDNF-EGFP转染BMSCs后,实时定量聚合酶联反应(real-time quantitativepolymerase chain reaction, RQ-PCR)和2-△△CT方法分析两目的基因HSV1-TK与BDNF的相对表达量(CT),并进行相关性分析；Western blot检测并分析HSV1-TK与BDNF的蛋白水平表达。
3.131I-FIAU的标记及BMSCs转染不同载体后对131I-FIAU的摄取
3.1.131I-FIAU的标记及质量鉴定
利用Iodogen固相氧化法对FAU进行放射性碘标记,标记产物用Sep-Pak-C-18反相层析色谱小柱进行纯化及标记率分析,TLC-SG硅胶板薄层层析法测定标记产物的放射化学纯度,并观察131I-FIAU在37℃正常人新鲜血清中放置4h、12h和24h后的体外稳定性。
3.2.不同载体转染BMSCs后对mI-FIAU的摄取研究
不同载体转染BMSCs 48h后,加入纯化后的131I-FIAU,在不同时间分别计数细胞培养基上清液中的放射性计数和消化后细胞的放射性计数,细胞摄取结果为：摄取率=C细胞悬液/(C细胞悬液+C细胞培养基)×100%。
第一组实验,重组真核质粒表达载体pcDNA3.1-TK-IRES-BDNF转染BMSCs 48h后,加入纯化后的131I-FIAU。在一定观察时间内,分析转染重组质粒的BMSCs对131I-FIAU的摄取数值随时间的变化,以转染质粒pcDNA3.1+为对照组。
第二组实验,在MOI分别为O、50、100、150、200、250转染BMSCs 48h后,加入纯化后的131I-FIAU。在相同时间下分析不同MOI的重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP转染BMSCs对131I-FIAU的摄取研究。
第三组实验,结合上述实验不同MOI的重组腺病毒Ad5-TK-IRES-BDNF-EGFP对BMSCs的转染效率,MTT对增殖能力的分析结果,不同MOI的重组腺病毒在相同时间下对131I-FIAU的摄取结果。以最佳MOI的重组腺病毒Ad5-TK-IRES-BDNF-EGFP转染BMSCs,分析转染重组腺病毒的BMSCs对131I-FIAU的摄取数值随时间变化的关系,以转染无效病毒Ad5-EGFP为对照组。
4.统计学方法
数据分析采用SPSS11.0软件进行,线性相关采用Pearson分析,两样本均数比较采用双侧t检验,以P＜0.05为有统计学意义。
结果
1.不同重组载体的构建和SD大鼠BMSCs的培养及鉴定
1.1.重组真核质粒表达载体pcDNA3.1-TK-IRES-BDNF的构建及鉴定
重组真核质粒表达载体pcDNA3.1-TK-IRES-BDNF经PCR、酶切和序列分析鉴定与预期设计一致。
1.2.重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP的构建及鉴定
由上海鸣鸿生物有限公司协作构建的重组表达质粒pDC316-TK-IRES-BDNF-EGFP经PCR、酶切鉴定和测序鉴定与预期设计一致；包装生成的重组腺病毒Ad5-TK-IRES-BDNF-EGFP的活病毒数量达到2.0×1010 PFU (plaque formation unit)/ml,对照重组腺病毒Ad5-EGFP的活病毒数量为3.6×1010PFU/ml。
1.3.大鼠BMSCs的培养及鉴定
采用密度梯度离心法获得到的BMSCs传到3-4代后,可获得较高纯度和保持良好的增殖能力；细胞免疫化学鉴定,CD44在95%以上的BMSCs表面表达,而CD34则未见明显表达。
2.不同载体转染体外培养的BMSCs实验研究
2.1.不同载体对BMSCs转染效率的测定
以阳离子脂质体(μl)与重组质粒载体(μg) pcDNA3.1-EGFP在2.5：1时转染BMSCs,48h时EGFP有较高表达效率,在随后的观察时间内EGFP的表达开始下降；不同MOI的重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP转染BMSCs后,绿色荧光显微镜下观察,在24h开始表达EGFP,48h达到高峰,120h开始下降；MOI为150时在转染效率可大于90%。
2.2.不同载体转染BMSC后细胞增殖能力、向神经元样细胞分化能力的检测
不同载体转染BMSCs后,在一定时间内于倒置显微镜下观察。发现重组质粒载体pcDNA3.1-TK-IRES-BDNF转染BMSCs后细胞密度明显减低,有较多细胞漂浮,台盘兰染色证实为死亡细胞。而重组腺病毒Ad5-TK-IRES-BDNF-EGFP的MOI为250时转染BMSCs后,在观察时间内细胞形态和密度均无明显变化,细胞培养基中仅可见极个别漂浮细胞。MTT比色法亦证实重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP对BMSCs的毒性作用较小, MOI在150时细胞存活率仍大于98%。重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP转染BMSCs的同时在细胞培养基中加入bFGF和EGF,倒置显微镜下观察细胞形态变化,发现转染重组腺病毒组和未转染重组腺病毒组的BMSCs均保持向神经元样细胞的良好分化能力,两组相比无明显差别。
2.3.不同载体转染BMSCs目的基因表达的检测
重组真核质粒表达载体pcDNA3.1-TK-IRES-BDNF转染BMSCs 48h后,通过RT-PCR技术可检测到HSV1-TK的表达,Western blot检测到BDNF的表达。不同MOI的重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP转染BMSCs后,通过RQ-PCR检测到两目的基因的表达,并且随着病毒MOI的增加表达增强。HSV1-TK和BNDF两目的基因的表达有良好的线性相关(r=0.973, p<0.05, n=3)。Western blot亦检测到了HSV1-TK和BNDF两目的基因的表达,随着病毒MOI的增加,基因表达也在增加。
3.131I-FIAU的标记及不同载体转染BMSCs后对131I-FIAU的摄取研究
3.1.131I-FIAU的标记及质量鉴定
Iodogen固相氧化法能有效标记FAU；经测定,标记物的标记率为(64.35±4.89)%(n=5)。TLC-SG硅胶板层析法测定,131I-FIAU的放射化学纯度为(98.62±0.62)%(n=5).131I-FIAU在正常人血清中37℃中孵育24h后,放射化学纯度仍大于95%。
3.2.不同载体转染BMSCs后对131I-FIAU的细胞摄取研究
第一组实验,重组质粒载体pcDNA3.1-TK-IRES-BDNF转染BMSCs后对131I-FIAU的摄取研究,以转染质粒pcDNA3.1+为对照组。
重组质粒载体pcDNA3.1-TK-IRES-BDNF转染BMSCs 48h后,加入纯化后的131I-FIAU,分析5h内对131I-FIAU的摄取。研究发现在前3h随着时间的延长摄取增加,在3h对131I-FIAU的摄取达到平台期,值为(4.15±0.074)%,n=3,在随后的观察时间内摄取数值增加不明显。在各个观察时间点转染目的质粒组与对照质粒组对131I-FIAU的摄取数值相比,有统计学意义(t=9.29-232.75,p<0.05,n=3)。
第二组实验,MOI分别为0、50、100、150、200、250的重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP转染BMSCs后,分析相同时间对131I-FIAU的细胞摄取。
不同MOI的重组腺病毒Ad5-TK-IRES-BDNF-EGFP转染BMSCs 48h后,在同一时间点3h时分析转染细胞对131I-FIAU的摄取。研究发现重组腺病毒随着MOI的增加对131I-FIAU的摄取增加,在MOI为150时达到平台期,随后随着MOI的增加转染重组腺病毒的BMSCs对131I-FIAU摄取增加不明显。
第三组实验中,相同MOI重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP转染BMSCs后,分析转染BMSCs不同时间对131I-FIAU的摄研究,以转染对照病毒Ad5-EGFP为对照组。
结合上述转染效率、MTT等实验结果,确定重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP的MOI为150时是对BMSCs最佳转染效率。MOI为150转染BMSCs 48h后,加入纯化后的131I-FIAU,分析不同时间转染重组腺病毒的BMSCs对131I-FIAU的摄取研究。结果发现在前3h内,转染了重组腺病毒Ad5-TK-IRES-BDNF-EGFP的BMSCs随着时间的延长,其摄取131I-FIAU也在增加,在3h摄取数值可达(31.42±0.46)%(n=3),此后随着时间的延长,摄取数值增加不明显。在观察时间内各时间点转染重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP的BMSCs对131I-FIAU的摄取均显著高于对照组,两组相比具有统计学意义(t=23.06-173.83,P<0.05,n=3)。
结论
本研究通过体外比较携带报告基因HSV1-TK和治疗基因BDNF的不同载体转染BMSCs实验研究,证实重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP是一种可以携带较大容量碱基,对BMSCs有较高转染效率,转染重组腺病毒后的BMSCs仍保持良好的增殖能力和向神经元样细胞诱导分化能力,IRES介导上游报告基因的表达可间接反映下游治疗基因的表达,二者具有良好的线性相关。利用Indogen固相氧化法对FAU进行放射性核素131I标记,标记产物用Sep-Pak-C-18反相层析色谱小柱进行纯化,是一种简单的标记及纯化技术,可得到较高的放射化学纯度。重组腺病毒载体Ad5-TK-IRES-BDNF-EGFP转染BMSCs可表达有活性的HSV1-TK,能有效介导BMSCs摄取131I-FIAU,在一定的MOI范围内其摄取数值随着MOI的增加而增加,有明显的剂量依赖性；此外,在一定的观察时间内随着时间的延长,对131I-FIAU的摄取数值亦在增加,显著高于对照组。本研究为后续以重组腺病毒Ad5-TK-IRES-BDNF-EGFP为载体介导的放射性核素报告基因活体监测踪转基因BMSCs治疗脑梗死的干细胞有效归巢数量、分化和功能表达过程,以及对外源基因的表达部位和时间的活体评估奠定了良好的实验基础。
Purpose
Constructing recombinant eukaryotic plasmid expression vector pcDNA3.1-TK-IRES-BDNF and recombinant adenovirus vector Ad5-TK-IRES-BDNF-EGFP, to assess the multiplication capacity, differentiation potential, uptake of 131I-FIAU capacity of rat BMSCs transfected with recombinant vectors, finally selecting a appropriate vector for further monitoring the genetically modified BMSCs in vivo
Methods
1. Construct different vectors and culture rat BMSCs
1.1. Construct and identify vector pcDNA3.1-TK-IRES-BDNF
The primers of HSV1-TK, IRES and BDNF were designed and sythesised according to Gene bank. Restriction enzyme sites were added into the both ends, which were matched with the multiple cloning sites (MCS) of eukaryotic expression plasmid vector pcDNA3.1+. The cDNA of target genes HSV1-TK and IRES were obtained from recombinant plasmid pcDNA3.1-TK, pIRES-EGFP by polymerase chain reaction (PCR) separately. The cDNA of target gene BDNF was obtained from a week-old SD rat brain tissue by RT-PCR technology. Three target genes were restricted with respective enzymes, and subcloned into the eukaryote plasmid pcDNA3.1+restricted with the same enzymes. Restriction analysis and sequencing were used to confirm the recombinant plasmid vector pcDNA3.1-TK-IRES-BDNF.
1.2. Construct and identify vector Ad5-TK-IRES-BDNF-EGFP
The plasmid vector, pDC316-TK-IRES-BDNF-EGFP, and the virus vector, E1/E3-deleted replication-defective recombinant adenovirus type 5 (Ad5-TK-IRES-BDNF-EGFP), carrying the HSV1-TK-IRES-BDNF coupling gene under the transcriptional control of the cytomegalovirus (CMV) promoter, were constructed and purified in the Vector Gene Technology Company Ltd. in Shanghai.
1.3. Culture rat BMSCs
The femur and tibia were isolated from health SD rat aged 4-6 weeks, the bone marrow were washed out with DMEM-F12 containg 15%FBS, and operated into the cultured bottles after density gradient centrifugation. BMSCs were cultivated and expanded in vitro. The cell morphology were observed under inverted microscope at different times; expression of surface antigens CD44 and CD34 were detected by immunocytochemistry.
2. BMSCs were transfected with different recombinant vectors in vitro
2.1. The transfection efficiency for BMSCs with different vectors
BMSCs were transfected with recombinant plasmid vector pcDNA3.1-EGFP, enhanced green fluorenscent protein (EGFP) expression were observed under fluorescence microscope, transfection efficiency was analyzed with flow cytometry (FCM). BMSCs were transfected with recombinant vector Ad5-TK-IRES-BDNF-EGFP at different multiplicity of infection (MOI), from 0,50,100,150,200 and 250, transfection efficiency was assayed by fluorescence microscope.
2.2. The proliferation and differentiation of genetically moditified BMSCs with different vectors
After BMSCs were transfected with different vectors, morphology and density changes of genetically moditified BMSCs were observed under inverted microscope at different times. MTT were used to detect the proliferation, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were added into the genetically moditified BMSCs, the morphology and the potential of differentiation were observed under inverted microscope.
2.3. The expression of target genes
When BMSCs were transfected with plasmid vector pCDNA3.1-TK-IRES-BDNF, RT-PCR and Western blot analysis were used to detect the expression of target genes in BMSCs. Real-time quantitative polymerase chain reaction (RQ-PCR) and Western blot were used to detect the expression of target gene in BMSCs transfected with different MOI of recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP.
3. The radioiodination of FAU and uptake 131I-FIAU for BMSCs transfected with different vectors
3.1. The radioiodination of FAU
Using solid phase oxidation with Iodogen, FAU was labeled with 131I. The product, 131I-FIAU, was purified on a reverse-phase Sep-Pak C-18 column and the labeling efficiency was then assayed. Moreover, the peak fractions were identified for the radiochemical purity assessment. To study the stability in vitro, the 131I-FIAU was incubated in serum at 37℃for 4,12 and 24 hours before the aliquots were removed for radiochemical purity analysis.
3.2. Uptake of 131I-FIAU for transgenic BMSCs by gamma counter
BMSCs were transfected with different vectors,48h later, the purified 131I-FIAU were added into the genetically modified BMSCs. The radiocounting of cell culture medium and digested cell were counted by gamma counter separately, uptake rate= C digested cell/(C digested cell+C cell culture medium)×100%.
PartⅠ, BMSCs were transfected with recombinant eukaryotic plasmid vector pcDNA3.1-TK-IRES-BDNF,48h later, adding purified 131I-FIAU. Uptake of 131I-FIAU was observed in a certain period of time, the control group was pcDNA3.1-EGFP.
PartⅡ, BMSCs were transfected with recombinant adenovirus vector Ad5-TK-IRES-BDNF-EGFP, the MOI was 0,50,100,150,200 and 250,48h later, adding the purified 131I-FIAU. Uptake of 131I-FIAU was observed at the same time, control group was Ad5-EGFP.
PartⅢ, the best MOI was obtained from the above-mentioned experiments, and then BMSCs were transfected with recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP in the best MOI. Uptake of 131I-FIAU was observed in a certain period of time, the control group was Ad5-EGFP.
4. Statistical methods
Data were pcocessed by SPSS 11.0 software.
Results
1. Construct different vectors and culture rat BMSCs
1.1. Construct and identify plasmid vector pcDNA3.1-TK-IRES-BDNF
The identification of recombinant eukaryotic expression plasmid vector pcDNA3.1-TK-IRES-BDNF by PCR, enzyme digestion and sequence analysis, and the result was matching with expectations.
1.2. Construct and identify vector Ad5-TK-IRES-BDNF-EGFP
The recombinant plasmid, pcDNA3.1-TK-IRES-BDNF, were identified by PCR、restriction enzyme digestion and sequencing. The titer of packaged recombinant adenovirus, Ad5-TK-IRES-BDNF-EGFP, was 2.0×1010 PFU/ml; A replication-defective adenovirus type 5 (Ad5-EGFP) was used as a control with the titer of 3.6×1010PFU/ml.
1.3. Culture and identify rat BMSCs
BMSCs can obtain high purity and good proliferation by gradient centrifugation. The surface antigen CD44 were observed in more than 95% of the BMSCs, while CD34 were no observed by immunohistochemistry.
2. BMSCs were transfected with different recombinant vectors in vitro
2.1. The transfection efficiency for BMSCs with different vectors
There was high transfection efficiency for BMSCs transfected with recombinant plasmid pcDNA3.1-EGFP, when the ratio between liposome (μl) and the recombinant plasmid (μg) was 2.5:1. When the MOI was 150, transfection efficiency for BMSCs transfected with recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP can reach peak, at the 48h, the transfection efficiency was more than 90%.
2.2. The proliferation and differentiation of transgenic BMSCs with different vectors
BMSCs transfected with recombinant plasmid pcDNA3.1-TK-IRES-BDNF, the cell density were increased seriously and there have many floating cells, death cells were confirmed by placenta blue staining. BMSCs transfected with recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP, the MOI was 0,50,100,150,200 and 250 separately. Little floating cell were observed under inverted microscope, when the MOI was 250. There have little toxicity for BMSC transected with recombinant adenovirus by MTT assay. The cell survival rate was more than 98% when the MOI was 150.
After BMSCs were transfected with recombinant adenovirus vector Ad5-TK-IRES-BDNF-EGFP, non-transfected recombinant adenovirus group was control, adding bFGF and EGF separately. The morphology change of genetically moditified BMSCs were observed under inverted microscope in a certain period of time, and the difference was no founded between adenovirus transfection group and non-transfected recombinant adenovirus group. Transgenic BMSCs remain fine ability of differentiation into neuron-like cells.
2.3. The expression of target gene were detected
The expression of TK and BDNF were detected with RT-PCR and Western blot separately, when BMSCs were transfected with recombinant eukaryotic expression vector pcDNA3.1-TK-IRES-BDNF,48h later. BMSCs were transfected with different MOI, the mRNA of TK and BDNF were deteted by RQ-PCR, expression of target gene upgrade with the increased MOI for a range. There was a strong relation between the mRNA expression of TK and BDNF for transfected BMSCs(r=0.973,p<0.05, n=3). The expression of target gene was detected by Western blot also.
3. The radioiodination of FAU and uptake 131I-FIAU for BMSCs transfected with different vectors
3.1. Radiolabeling and stability
FAU could be labeled efficiently by solid phase oxidation with Iodogen. The radiolabeling efficiency of 131I-FIAU was (64.35±4.89)%(n=5). After purification on Sep-Pak C-18 column, the radiochemical purity of the final product 131I-FIAU was (98.62±0.62)%(n=5). The radiochemical purity of 131I-FIAU remained above 95% after incubated in serum at 37℃for 4,12 and 24 hours, which indicated 131I-FIAU was stable in serum.
3.2. Uptake of 131I-FIAU for genetically modified BMSCs by gamma counter
PartⅠ, BMSCs were transfected with recombinant eukaryotic plasmid vector pcDNA3.1-TK-IRES-BDNF,48h later, adding purified 131I-FIAU. Uptake of 131I-FIAU was observed in 5 hours. Uptake of 131I-FIAU for genetically modified BMSCs was increased with time extension in 3 hours, there has significant increase compared with the control.
PartⅡ, BMSCs were transfected with recombinant adenovirus vector Ad5-TK-IRES-BDNF-EGFP, the MOI was 0,50,100,150,200 and 250 separately,48h later, adding the purified 131I-FIAU. Uptake of 131I-FIAU was observed in 3 hours. Uptake of 131I-FIAU for genetically modified BMSCs was increased with the MOI raising, uptake was reach peak at 3h point, when the MOI was 150.
PartⅢ, the best MOI was obtained from the above-mentioned experiment, the best MOI was 150. Then BMSCs were transfected with recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP at MOI was 150. Uptake of 131I-FIAU was observed in 5 hours, uptake of 131I-FIAU for genetically moditified BMSCs was increased with time extension in 3 hours. There has significance between genetically modified BMSCs and control BMSCs for uptake of131I-FIAU at all the time points(t=20.74-98.16, p<0.05, n=3).
Conclusion
Recombinant adenovirus can contain more bases, get higher transfection efficiency, lower toxic for BMSCs, and remain fine differentiation into neuron-like cells. There has a strong relation between the mRNA expression of HSV1-TK and BDNF in genetically modified BMSCs. The expressions of upstream reporter gene can indirect response to the downstream therapeutic gene by IRES. FAU could be labeled efficiently by solid phase oxidation with Iodogen; the high radiochemical purity for product can obtain by Sep-Pak-C-18 reversed-phase chromatography. BMSCs transfected with recombinant adenovirus vector Ad5-TK-IRES-BDNF-EGFP can express HSV1-TK possessed fine activity, effective uptake of 131I-FIAU, there has significance between genetically modified BMSCs and control BMSCs for uptake of 131I-FIAU at all the time points. This study indicates that recombinant adenovirus Ad5-TK-IRES-BDNF-EGFP may be a suitable gene vector for research tracing genetically modified stem cells.
引文
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