PDE5-siRNA和iNOS基因修饰的脂肪干细胞在勃起功能障碍治疗中的体外研究
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摘要
目的通过比较研究PDE5-siRNA和iNOS单双基因在大鼠ADSCs中的表达效应,探讨PDE5-siRNA和iNOS基因修饰ADSCs应用于ED治疗的可行性和有效性。
方法采用Ⅰ型胶原酶消化法原代培养SD大鼠ADSCs,并通过流式细胞术检测间充质干细胞相关免疫表型和定向多向诱导分化鉴定ADSCs。取第4代ADSCs,分别采用10μmol/L氯甲基苯甲酰氨(CM-Dil)荧光活性染料和50μmol/L5-乙炔基-2’-脱氧尿嘧啶核苷(EdU)进行标记,分别于标记后第7天、14天、21天和28天通过流式细胞术和荧光显微镜比较研究CM-Dil和EdU对ADSCs的体外标记示踪效果。分别构建携带大鼠PDE5-siRNA的重组慢病毒载体(LV-PDE5-siRNA-GFP)和iNOS基因的重组腺病毒载体(Ad-iNOS-EGFP),并通过LV-PDE5-siRNA-GFP (MOI=70)和Ad-iNOS-EGFP (MOI=30)共转染第4代ADSCs,同时设立空白对照组(未转染组)、阴性对照组一(LV-NC-siRNA-GFP转染组)、阴性对照组二(Ad-NC-EGFP转染组)、阴性对照组三(LV-NC-siRNA-GFP+Ad-NC-EGFP共转染组)、阳性对照组一(LV-PDE5-siRNA-GFP转染组)、阳性对照组二(Ad-iNOS-EGFP转染组)。于共转染后3d、5d、7d、10d和14d,分别采用实时荧光定量PCR和Western Blot技术检测PDE5基因和iNOS基因在mRNA水平和蛋白水平的表达情况。同时,分别采用硝酸还原酶法和酶联免疫吸附法(ELISA)测定ADSCs中NO和cGMP浓度水平。
结果培养的细胞阳性表达间充质干细胞免疫表型CD49d、 CD73、CD90、CD105和CD106而CD31、CD34和CD45为阴性表达,且成脂肪诱导分化细胞油红O染色阳性、成平滑肌诱导分化细胞共表达a-SMA和Desmin蛋白、成血管内皮诱导分化细胞vWF表达为阳性、成神经诱导分化细胞Nestin和NeuN表达阳性,而对照组均为阴性。实验组(LV-PDE5-siRNA-GFP+Ad-iNOS-EGFP共转染组)PDE5基因在mRNA水平和蛋白水平于共转染后3d明显下调,5-7d PDE5-siRNA干扰效率达到峰值,10d后开始降低,但第14天干扰效率仍维持较高水平,与阳性对照组-(LV-PDE5-siRNA-GFP转染组)间差异无统计学意义(P>0.05)；同时,腺病毒介导的iNOS基因于共转染24h后在mRNA水平和蛋白水平均显著上调,并于转染后5-7d达到峰值,10d后开始降低,14天后仍维持较高表达水平,其表达水平与阳性对照组二(Ad-iNOS-EGFP转染组)间差异无统计学意义(P>0.05)。共转染后3d、5d、7d、10d和14d,实验组(LV-PDE5-siRNA-GFP+Ad-iNOS-EGFP共转染组)和阳性对照组二(Ad-iNOS-EGFP转染组)NO浓度于转染后3d开始增加,5-7d达到峰值,于第10天出现降低后再呈增加趋势,至第14天仍维持较高浓度水平,且两组间差异无统计学意义(P>0.05)；实验组(LV-PDE5-siRNA-GFP+Ad-iNOS-EGFP共转染组)、阳性对照组一(LV-PDE5-siRNA-GFP转染组)和阳性对照组二(Ad-iNOS-EGFP转染组)cGMP浓度亦于转染后3d显著增加,5～7d达到峰值,于第10天后出现降低后再呈增加趋势,至第14天仍维持较高浓度水平,而实验组cGMP浓度显著高于两阳性对照组(P<0.01),但两阳性对照组间差异无显著统计学意义(P>0.05)。
结论PDE5-siRNA和iNOS基因于ADSCs中同时对PDE5和iNOS基因表达发挥调控作用,并同时有效持续提高细胞内NO和cGMP浓度水平。因此,PDE5-siRNA和iNOS基因修饰的ADSCs于CM-Dil标记后有望在基因水平和组织学水平达到治疗ED目的,且将具有比无／单基因修饰的ADSCs更显著的疗效。
第一部分大鼠脂肪干细胞的培养和鉴定
目的体外分离培养大鼠ADSCs并进行鉴定,为PDE5-siRNA和iNOS基因修饰ADSCs移植治疗ED提供良好的种子细胞。
方法取SD大鼠腹股沟区皮下脂肪,采用Ⅰ型胶原酶消化法获取大鼠ADSCs,行传代培养,并取第4代细胞行CCK-8法测定细胞生长曲线。流式细胞术检测间充质干细胞免疫表型CD31、CD34、CD45、CD49d、 CD73、CD90、CD105和CD106表达情况初步鉴定ADSCs,再于成脂肪细胞、成平滑肌细胞、成血管内皮细胞和成神经细胞诱导培养基多向诱导分化不同时间后通过油红O染色、α-平滑肌肌动蛋白(a-SMA)和结蛋白(Desmin)、血管性血友病因子(vWF)、神经元核抗原(NeuN)和巢蛋白(Nestin)免疫荧光检测对诱导分化细胞进行鉴定。
结果第4代大鼠ADSCs生长曲线呈“S”形,为标准的细胞生长曲线。所培养的细胞阳性表达间充质干细胞相关阳性免疫表型CD49d、CD73、CD90、CD105和CD106,阳性率分别为(1.88±0.12)%、(4.05±0.13)%、(99.07±0.45)%、(4.68±0.23)%和(11.81±3.60)%,而造血干细胞表面分子CD34、CD45和血管内皮细胞表面分子CD31均为阴性表达,阳性率分别为(0.35±0.04)%、(0.38±0.01)%和(0.54±0.33)%。多向诱导分化鉴定显示,成脂肪诱导分化细胞油红O染色阳性,成平滑肌诱导分化细胞α-SMA和Desmin蛋白表达呈强阳性,成血管内皮诱导分化细胞vWF为阳性,成神经诱导分化细胞Nestin和NeuN表达阳性,对照组均为阴性,表明所培养的细胞具备多向诱导分化潜能。
结论成功从大鼠脂肪组织中分离培养ADSCs,为PDE5-siRNA和iNOS基因修饰干细胞提供了理想的细胞载体,从而为PDE5-siRNA和iNOS基因修饰ADSCs移植治疗ED提供良好的种子细胞。
第二部分大鼠脂肪干细胞的体外标记和示踪
目的比较研究氯甲基苯甲酰氨(CM-Dil)荧光活性染料和5-乙炔基-2’-脱氧尿嘧啶核苷(EdU)对大鼠ADSCs的体外标记示踪效果,为ADSCs移植治疗ED寻找最佳的干细胞示踪技术。
方法取第4代大鼠ADSCs,分别采用2μmol/L、4μmol/L、6μmol/L、8μmol/L、10μmol/L、12μmol/L、14μmol/L、16μmol/L、18μmol/L和20μmol/L CM-Dil不同标记浓度进行标记,通过流式细胞术和荧光显微镜筛选最佳标记浓度,并于最佳浓度标记后7d、14d、21d、28d和35d流式细胞术检测细胞阳性率及荧光强度。同时,50μmol/L EdU培养基分别标记ADSCs2h6^、12h、24h、48h、72h和96h,通过显色反应后于荧光显微镜下观察EdU标记不同时间的细胞阳性率,再于最佳标记时间标记后7d、14d、21d和28d检测EdU标记细胞阳性率。
结果CM-Dil标记浓度达10μmol/L时,ADSCs即发生饱和,标记效率即可近达100%,且该浓度对ADSCs的生长状态无明显影响。10μmol/L CM-Dil于标记后第7天和第14天细胞阳性率较高,组间差异无统计学意义(P>0.05)；第21天后荧光逐渐衰减,细胞阳性率开始降低(P<0.05),第28天明显降低但阳性率仍达80%以上。50μmol/L EdU标记ADSCs48h其标记效率达到峰值为最佳标记时间,EdU标记后第7天细胞阳性率开始降低(P<0.01),第14天急剧下降,第28天细胞阳性率仅为(14.39±2.67)%。
结论CM-Dil和EdU两者均可用于ADSCs标记、示踪,但CM-Dil标记具有操作简单、标记阳性率高、荧光持久等优点,适用于长期标记示踪,EdU仅适用于短期标记示踪。因此,CM-Dil标记示踪为ADSCs移植治疗ED的理想示踪技术。
第三部分
PDE5-siRNA和iNOS基因重组病毒载体的构建和鉴定
一、PDE5-siRNA重组慢病毒载体的构建
目的通过构建携带大鼠PDE5-siRNA的重组慢病毒载体(LV-PDE5-siRNA-GFP),为PDE5-siRNA植入ADSCs并稳定发挥干扰效应提供良好媒介。
方法直接选用课题组前期研究有效的PDE5-siRNA序列(GCAGCCGAATTCTT-TGATC),并合成含有靶序列的双链DNA Oligo。采用限制性内切酶Hpa I和Xho I对pFU-GW-RNAi慢病毒载体进行双酶切使其线性化,再通过T4DNA连接酶将双链DNA Oligo与线性化的pFU-GW-RNAi慢病毒载体连接并转化大肠杆菌DH5a感受态细胞,然后单克隆菌落采用PCR法鉴定,PCR鉴定阳性的克隆进一步行测序鉴定和比对分析,比对正确的克隆即为构建正确的目的载体。将pFU-GW-PDE5-siRNA重组慢病毒载体、pHelper1.0和pHelper2.0'慢病毒辅助包装质粒三质粒共转染293T细胞,转染48h后,收集293T细胞上清,然后进行病毒扩增、浓缩和纯化,最后采用孔稀释法测定慢病毒滴度,即病毒滴度(TU/mL)=最后一孔GFP阳性细胞数/病毒原液量×(1E+3)。
结果测序拼接序列中含有PDE5-siRNA序列(GCAGCCGAATTCTTTGATC)且匹配率为100%。1E+1μL、1E+OμL、1E-1μL、1E-2μL、1E-3μL、1E-4μL、1E-5μL1E-6μL和1E-7μL LV-PDE5-siRNA-GFP重组慢病毒稀释液转染293T细胞48h后,1E+1μL-1E-6μL转染孔均表达GFP,且GFP阳性细胞数随稀释倍数的增加而递减,1E-6μL转染孔仅检测到1个GFP阳性细胞,因此该病毒滴度为1E+9TU/mL。
结论携带大鼠PDE5-siRNA的重组慢病毒载体构建成功且获得较高病毒滴度,可用于ADSCs的基因修饰研究。
二、iNOS基因重组腺病毒载体的构建和鉴定
目的通过构建携带增强型绿色荧光蛋白(EGFP)和大鼠诱导型一氧化氮合酶(iNOS)基因的重组腺病毒载体,并观察由腺病毒介导的iNOS基因在293T细胞中的表达情况,为模拟iNOS基因在ADSCs中的表达提供可靠依据。
方法采用PCR方法钓取和扩增大鼠iNOS基因并获取iNOS基因全长片段。通过Age I限制性内切酶酶切pDC315-EGFP腺病毒载体使其线性化。通过In-Fusion交换酶将纯化的iNOS基因PCR产物与线性化的pDC315-EGFP腺病毒载体进行定向克隆连接,将连接产物转化感受态细胞,并采用PCR法鉴定单克隆菌落,对于PCR鉴定阳性的克隆进一步行测序鉴定和BLAT比对分析。将构建正确的pDC315-iNOS-EGFP质粒转染293T细胞,并于转染后48h采用Western Blot技术检测iNOS基因在293T细胞中的表达情况。成功构建的pDC315-iNOS-EGFP质粒,利用AdMax腺病毒包装系统包装产生重组腺病毒,病毒粗提液反复感染HEK293细胞行病毒扩增,病毒粗提液通过Adeno-X腺病毒纯化试剂盒进行纯化,最后采用倍比稀释法测定病毒滴度。取不同体积病毒原液转染293T细胞,于转染48h后通过Western Blot技术检测iNOS基因在蛋白水平的表达情况。
结果经PCR鉴定、限制性酶切分析、测序鉴定和目的质粒Western Blot表达检测鉴定,证实pDC315-iNOS-EGFP重组腺病毒载体构建成功。重组腺病毒包装成功,且病毒滴度为1.2E+10PFU/mL。iNOS基因重组腺病毒转染293T细胞48h后,Western Blot检测到约154KDa大小的条带,其与iNOS/GFP融合蛋白分子量大小基本相一致,表明腺病毒介导的iNOS基因成功在293T细胞中正确表达。
结论iNOS基因重组腺病毒载体构建成功且获得较高病毒滴度,为iNOS基因修饰ADSCs移植治疗ED奠定了良好的前期实验基础。
第四部分
PDE5-siRNA和iNOS基因在大鼠脂肪干细胞中的表达研究
目的观察PDE5-siRNA和iNOS基因在大鼠ADSCs中的表达效应,探讨PDE5-siRNA和iNOS基因修饰ADSCs移植治疗ED的可能性和有效性。
方法LV-PDE5-siRNA-GFP (MOI=70)和Ad-iNOS-EGFP (MOI=30)共转染第4代ADSCs,同时设立空白对照组(未转染组)、阴性对照组一(LV-NC-siRNA-GFP转染组)、阴性对照组二(Ad-NC-EGFP转染组)、阴性对照组三(LV-NC-siRNA-GFP+Ad-NC-EGFP共转染组)、阳性对照组-(LV-PDE5-siRNA-GFP转染组)和阳性对照组二(Ad-iNOS-EGFP转染组)。共转染后3d,采用Annexin V-PE/7-AAD凋亡试剂盒检测ADSCs凋亡情况,并于共转染第3天、5天、7天、10天和14天,分别采用实时荧光定量PCR和Western Blot技术检测PDE5和iNOS基因在mRNA水平和蛋白水平的表达情况。同时,分别采用硝酸还原酶法和ELISA法测定ADSCs培养上清中NO浓度和细胞内cGMP浓度。
结果实验组(LV-PDE5-siRNA-GFP+Ad-iNOS-EGFP）共转染组凋亡细胞比例为(3.04+0.58)%,与其他各组间差异无显著统计学意义(P>0.05)。实验组PDE5基因在mRNA水平和蛋白水平于转染后3d明显下调,5-7d PDE5-siRNA干扰效率达到峰值,10d后开始降低,但第14天干扰效率仍维持较高水平,与阳性对照组一差异无统计学意义(P>0.05)；腺病毒介导的iNOS基因于转染24h后在mRNA水平和蛋白水平出现显著上调,转染后5-7d达到峰值,10d后开始降低,14天后仍维持较高表达水平,其表达水平与阳性对照组二差异无统计学意义(P>0.05)；细胞内NO和cGMP浓度于转染3d后显著增加,5-7d达到峰值,于第10天出现明显降低后再次增加,至第14天仍维持较高浓度水平。
结论PDE5-siRNA和iNOS基因在ADSCs中有效、稳定共表达,且能同时有效提高细胞内NO和cGMP浓度水平,从而为PDE5-siRNA和iNOS基因修饰的ADSCs移植治疗ED的体内研究提供了有力的工具。
Objective To study the effects of rat PDE5gene specific small interference RNA (PDE5-siRNA) and iNOS single/double gene expression in rat adipose-derived stem cells (ADSCs), and investigate the feasibility and effectiveness of ADSCs modified with PDE5-siRNA and iNOS gene for erectile dysfunction (ED) therapy.
Methods Isolate and culture ADSCs from the adipose tissue of rat inguinal region by collagenase digestion. The cultured cells were identified not only by flow cytometer detection of mesenchymal stem cells (MSCs) related-immunophynotypes, but also by their capacities in the adipogenic, myogenic, endothelial and neurogenic differentiation. Then the fourth passage ADSCs were labeled with10μmol/L chloromethyl-benzamidodialkyl-carbocyanine (CM-Dil) and50μmol/L5-ethynyl-2'-deoxyuridine (EdU), and the labeling effects of CM-Dil and EdU on ADSCs were detected by flow cytometer and fluorescence microscope at7days,14days,21days and28days, respectively. The recombinant lentivirus vector expressing PDE5-siRNA (LV-PDE5-siRNA-GFP) and recombinant adenovirus vector carrying iNOS gene (Ad-iNOS-EGFP) were constructed, respectively. PDE5-siRNA and iNOS gene were cotransfected into ADSCs mediated by lentivirus and adenovirus. The experimental groups were designed as blank group, negative control group1(LV-NC-siRNA-GFP transfected group), negative control group2(Ad-NC-EGFP transfected group), negative control group3(LV-NC-siRNA-GFP and Ad-NC-EGFP cotransfected group), positive control group1(LV-PDE5-siRNA-GFP transfected group) and positive control group2(Ad-iNOS-EGFP transfected group) and experimental group (LV-PDE5-siRNA-GFP and Ad-iNOS-EGFP cotransfected group). The expression of PDE5and iNOS gene were examined with real-time fluorescence quantitative RT-PCR assay and western blot at3days,5days,7days,10days and14days, respectively. At the same time, the concentration of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) were assayed by nitrate reductase method and enzyme-linked immunosorbent assay (ELISA).
Results The cultured cells expressed CD49d, CD73, CD90, CD105and CD106, lacked CD31, CD34and CD45. Adipogenic, myogenic, endothelial and neurogenic differentiation cells can be stained with Oil Red O, a-SMA and Desmin, vWF, NeuN and Nestin, respectively, and the negative control was negative. The expression of PDE5gene was downregulated at3days, reached the peak at5to7days, and decreased at10days, and remained higher later. Compared to positive control group1(LV-PDE5-siRNA-GFP transfected group), no significant difference was noted (P>0.05). The mRNA and protein expression of iNOS gene mediated by recombinant adenovirus vector worked at24hours, reached a peak at5to7days, decreased at10days, and lasted at least2weeks. The expression level of PDE5was down-regulated by more than70percent from3to14days, significantly lower than negative control (P<0.001). However, there was no significant difference between cotransfected group and positive control group2(Ad-iNOS-EGFP transfected group), P>0.05. The NO concentration of cotransfected group and positive control group2was elevated at3days, reached the peak at5to7days, decreased at10days, and kept at least14days, but no greater than positive control group2(P>0.05). At the same time, the concentration of cGMP in cotransfected group and positive control groups increased at3days, reached the peak at5to7days, decreased at10days, but still kept at a high level later. The concentration level of cotransfected group was much higher than positive control groups (P<0.01), but no significant difference was noted between positive control group1and group2(P>0.05). The results showed that the concentration of NO and cGMP in ADSCs were elevated at3days, reached the peak at5to7days, decreased at10days but increased later, and lasted at least14days.
Conclusion PDE5-siRNA and iNOS gene mediated by lentivirus/adenovirus vector have successfully cotransfected and co-expressed in ADSCs. ADSCs modified by PDE5-siRNA and iNOS double gene could produce enough NO and more cGMP, which is more effective than single gene modified. Therefore, CM-Dil labeling ADSCs modified with PDE5-siRNA and iNOS genes could be the ideal seed cells in the future treatment of ED.
PartⅠ
Culture and Identification of Rat Adipose-Derived Stem Cells
Objective To isolate and culture rat ADSCs in vitro, and provide the available seed cells for PDE5-siRNA and iNOS gene modified stem cells in the future treatment of ED.
Methods Obtain adipose tissue from rat inguinal region by surgical resection, and isolate and culture ADSCs by collagenase type I digestion method. The fourth generation cell growth curve was described with cell counting kit-8(CCK-8). Mesenchymal stem cells (MSCs) related-immunophynotypes CD31, CD34, CD45, CD49d, CD73, CD90, CD105and CD106were detected by flow cytometer detection. The characterization of ADSCs was also arrayed by inducing in the adipogenic, myogenic, endothelial and neurogenic differentiation, and identified with Oil Red O staining, a-SMA and Desmin staining,vWF staining, NeuN and Nestin staining, respectively.
Results The growth curve of the fourth passage cultured cells was presented typical "S" shape. The cultured cells expressed CD49d, CD73, CD90, CD105and CD106, lacked CD31, CD34and CD45. The positive rate was (1.88±0.12)%,(4.05±0.13)%,(99.07±0.45)%,(4.68±0.23)%,(11.81±3.60)%,(0.35±0.04)%,(0.38±0.01)%and (0.54±0.33)%, respectively. Adipogenic, myogenic, endothelial and neurogenic differentiation cells can be stained with Oil Red O, a-SMA and Desmin, vWF, NeuN and Nestin, respectively. However, the negative control was negative.
Conclusion ADSCs have been successfully isolated and cultured from rat adipose tissue, and could be used as ideal cell carrier for PDE5-siRNA and iNOS gene modified stem cells, provide the available seed cells for PDE5-siRNA and iNOS gene modified stem cells in the future treatment of ED.
Part Ⅱ
Cell Tracker Labeling for Rat Adipose-Derived Stem Cells
Objective To compare the labeling effects of CM-Dil and EdU on rat ADSCs, and seek the optimal stem cell labeling technique applied to ED therapy.
Methods The fourth generation ADSCs were labeled with2μmol/L,4μmol/L,6μmol/L,8μmol/L,10μmol/L,12μmol/L,14μmol/L,16μmol/L,18μmol/L and20μmol/L CM-Dil solution. The optimal labeling concentration was screened by flow cytometer and fluorescence microscope. Then the CM-Dil positive cell rate was detected by flow cytometer and fluorescence microscope at7days,14days,21days and28days, respectively. In order to seek the optimal labeling time of EdU for ADSCs, ADSCs were labeled with50μmol/L EdU containing medium incubated for2h,6h,12h,24h,48h,72h and96h. EdU positive cell rate was detected by fluorescence microscope at7days,14days,21days and28days, respectively.
Results The optimal labeling concentration of CM-Dil for ADSCs was10μmol/L, and the positive cell rate was near to100%.10μmol/L CM-Dil had no bad effects on the growth of ADSCs. The positive cell rate was higher at7days and14days (P>0.05), decreased at21days (P<0.05), but still kept more than80%at28days. The labeling efficiency of EdU reached the peak incubating for48hours, and decreased at72hours. The positive rate of EdU labeling cells was (87.1±1.8)%, and decreased at7days (P<0.01), and the positive rate was only (14.4±2.7)%at28days.
Conclusion CM-Dil and EdU are suitable for ADSCs tracker labeling. However, CM-Dil gains much more advantages than EdU in long-term tracing, which may be an ideal and optimal ADSCs tracing technology in the future treatment of ED.
Part Ⅲ
Construction and Identification of Recombinant Viral Vector Carrying Rat PDE5-siRNA and iNOS Gene
PartⅢ-A
Construction of Recombinant Lentivirus Vector Expressing RatPDE5-siRNA
Objective Construct recombinant lentivirus vector expressing rat PDE5-siRNA (LV-PDE5-siRNA-GFP), and provide a good transfer media for PDE5-siRNA expression in ADSCs thereby.
Methods The effective interfere sequence for PDE5(GCAGCCGAATTCTTTGATC) was obtained from our prophase research results. Double-stranded DNA Oligo containing PDE5-siRNA sequence was synthesized and connected with linearized pFU-GW-RNAi recombinant lentivirus vector by T4DNA ligase. There recombinant pFU-GW-PDE5-siRNA recombinant lentivirus vector was transformed into competent cells. The positive recombination was selected by polymerase chain reaction (PCR) and further identified by sequencing analysis.293T cells were infected by successfully constructed pFU-GW-PDE5-siRNA lentivirus vector together with pHelper1.0and pHelper2.0plasmid. Cellular supernatant of transfected293T cells was harvested, concentrated and purified at48hours. The lentivirus titer was determined by dilution method.
Results The sequencing results showed that targeted PDE5-siRNA sequence GCAGCC-GAATTCTTTGATC was was consistent with pFU-GW-PDE5-siRNA lentivirus vector sequence. The lentivirus titer was1E+9TU/mL.
Conclusion Recombinant lentivirus vector carrying rat PDE5-siRNA was successfully constructed and high lentivirus titer was obtained, which could be applied to further study.
PartⅢ-B
Construction and Identification of Recombinant Adenovirus Vector Harboring Rat iNOS Gene
Objective Construct recombinant adenovirus over-expression vector harboring enhanced green fluorescent protein (EGFP) and rat iNOS gene, and observe iNOS gene expression mediated by recombinant adenovirus in293T cells, which may provide reliable basis for the possibility of iNOS gene expression in ADSCs.
Method The full-length iNOS gene was fished and amplified by PCR. pDC315-EGFP adenovirus vector was linearized with Age I digestion, then connected with iNOS gene. There recombinant pDC315-iNOS-EGFP adenovirus vector was transformed into competent cells and identified by restriction enzyme digestion, PCR and DNA sequencing.293T cells were transfected with confirmed pDC315-iNOS-EGFP plasmid. The expression of pDC315-iNOS-EGFP plasmid was detected by western blot assay. The recombinant adenovirus was generated by AdMax packaging system in HEK293cells. Raw adenovirus was harvested, concentrated and purified by Adeno-X Maxi purification kit. The lentivirus titer was determined by doubling dilution method. The iNOS gene recombinant adenovirus was identified by western blot in293T cells.
Results pDC315-iNOS-EGFP adenovirus vector was successfully constructed and confirmed by restriction enzyme digestion, PCR, DNA sequencing and western blot assay. The adenovirus titer was1.2E+10PFU/mL. The iNOS/GFP fusion proteins were successfully expressed in293T cells observing by western blot at48hours.
Conclusion Recombinant adenovirus over-expression vector harboring rat iNOS gene was successfully constructed and expressed in293T cells, which could be further applied to the modification of ADSCs.
Part IV
The Expression of PDE5-siRNA and iNOS Gene in Rat Adipose-Derived Stem Cells
Objective To compare the effects of rat PDE5-siRNA and iNOS single/double gene rat on ADSCs, and investigate the possibility and feasibility of ADSCs modified with PDE5-siRNAand iNOS gene for future ED therapy by grafting into corpus cavernosum.
Methods The experiment groups were designed as blank group, negative control group1(LV-NC-siRNA-GFP transfected group), negative control group2(Ad-NC-EGFP transfected group), negative control group3(LV-NC-siRNA-GFP and Ad-NC-EGFP cotransfected group), positive control group1(LV-PDE5-siRNA-GFP transfected group) and positive control group2(Ad-iNOS-EGFP transfected group) and experimental group (LV-PDE5-siRNA-GFP and Ad-iNOS-EGFP cotransfected group). ADSCs were cotransfected with LV-PDE5-siRNA-GFP (MOI=70) and Ad-iNOS-EGFP (MOI=30). The apoptosis of transfected ADSCs was detected by Annexin V-PE/7-AAD apoptosis kit and flow cytometry at3days. The expressions of PDE5and iNOS gene in ADSCs were detected by real time RT-PCR and western blot assay at3days,5days,7days,10days and14days. Nitric oxide and cGMP were determined by Nitrate/Nitrite Assay Kit cultured with lOmM L-arginine containing medium for24hours and ELISA Kit cultured with10μM sodium nitroprusside containing medium for2minutes, respectively.
Results The percentage of cotransfected ADSCs apoptosis was (3.04±0.58)%, and no significant difference was noted between the7groups (P>0.05). PDE5-siRNA worked at3days, reached a peak at5to7days, decreased at10days, and lasted at least14days. The expression level of PDE5was down-regulated by more than70percent from3to14days, significantly lower than negative control (P<0.001). iNOS gene began to express at24hours, reached a peak at4to5days, and decreased at10days. There was an overexpression of iNOS gene in ADSCs modified with iNOS (P<0.001). The level of NO was greatly higher than negative control (P<0.01), but no greater than iNOS single gene modified control (P>0.05). The concentration of cGMP was significantly greater than PDE5-siRNA/iNOS single gene modified control (P<0.01). The activation effects of PDE5-siRNA and iNOS double gene on NO/cGMP in ADSCs were kept at least2weeks.
Conclusion PDE5-siRNA and iNOS gene were coexpressed in ADSCs, and produced enough NO and more cGMP thereby. Therefore, ADSCs modified with PDE5-siRNA and iNOS genes could be the ideal seed cells in the future treatment of ED by grafting into corpus cavernosum.
方法采用Ⅰ型胶原酶消化法原代培养SD大鼠ADSCs,并通过流式细胞术检测间充质干细胞相关免疫表型和定向多向诱导分化鉴定ADSCs。取第4代ADSCs,分别采用10μmol/L氯甲基苯甲酰氨(CM-Dil)荧光活性染料和50μmol/L5-乙炔基-2’-脱氧尿嘧啶核苷(EdU)进行标记,分别于标记后第7天、14天、21天和28天通过流式细胞术和荧光显微镜比较研究CM-Dil和EdU对ADSCs的体外标记示踪效果。分别构建携带大鼠PDE5-siRNA的重组慢病毒载体(LV-PDE5-siRNA-GFP)和iNOS基因的重组腺病毒载体(Ad-iNOS-EGFP),并通过LV-PDE5-siRNA-GFP (MOI=70)和Ad-iNOS-EGFP (MOI=30)共转染第4代ADSCs,同时设立空白对照组(未转染组)、阴性对照组一(LV-NC-siRNA-GFP转染组)、阴性对照组二(Ad-NC-EGFP转染组)、阴性对照组三(LV-NC-siRNA-GFP+Ad-NC-EGFP共转染组)、阳性对照组一(LV-PDE5-siRNA-GFP转染组)、阳性对照组二(Ad-iNOS-EGFP转染组)。于共转染后3d、5d、7d、10d和14d,分别采用实时荧光定量PCR和Western Blot技术检测PDE5基因和iNOS基因在mRNA水平和蛋白水平的表达情况。同时,分别采用硝酸还原酶法和酶联免疫吸附法(ELISA)测定ADSCs中NO和cGMP浓度水平。
结果培养的细胞阳性表达间充质干细胞免疫表型CD49d、 CD73、CD90、CD105和CD106而CD31、CD34和CD45为阴性表达,且成脂肪诱导分化细胞油红O染色阳性、成平滑肌诱导分化细胞共表达a-SMA和Desmin蛋白、成血管内皮诱导分化细胞vWF表达为阳性、成神经诱导分化细胞Nestin和NeuN表达阳性,而对照组均为阴性。实验组(LV-PDE5-siRNA-GFP+Ad-iNOS-EGFP共转染组)PDE5基因在mRNA水平和蛋白水平于共转染后3d明显下调,5-7d PDE5-siRNA干扰效率达到峰值,10d后开始降低,但第14天干扰效率仍维持较高水平,与阳性对照组-(LV-PDE5-siRNA-GFP转染组)间差异无统计学意义(P>0.05)；同时,腺病毒介导的iNOS基因于共转染24h后在mRNA水平和蛋白水平均显著上调,并于转染后5-7d达到峰值,10d后开始降低,14天后仍维持较高表达水平,其表达水平与阳性对照组二(Ad-iNOS-EGFP转染组)间差异无统计学意义(P>0.05)。共转染后3d、5d、7d、10d和14d,实验组(LV-PDE5-siRNA-GFP+Ad-iNOS-EGFP共转染组)和阳性对照组二(Ad-iNOS-EGFP转染组)NO浓度于转染后3d开始增加,5-7d达到峰值,于第10天出现降低后再呈增加趋势,至第14天仍维持较高浓度水平,且两组间差异无统计学意义(P>0.05)；实验组(LV-PDE5-siRNA-GFP+Ad-iNOS-EGFP共转染组)、阳性对照组一(LV-PDE5-siRNA-GFP转染组)和阳性对照组二(Ad-iNOS-EGFP转染组)cGMP浓度亦于转染后3d显著增加,5～7d达到峰值,于第10天后出现降低后再呈增加趋势,至第14天仍维持较高浓度水平,而实验组cGMP浓度显著高于两阳性对照组(P<0.01),但两阳性对照组间差异无显著统计学意义(P>0.05)。
结论PDE5-siRNA和iNOS基因于ADSCs中同时对PDE5和iNOS基因表达发挥调控作用,并同时有效持续提高细胞内NO和cGMP浓度水平。因此,PDE5-siRNA和iNOS基因修饰的ADSCs于CM-Dil标记后有望在基因水平和组织学水平达到治疗ED目的,且将具有比无／单基因修饰的ADSCs更显著的疗效。
第一部分大鼠脂肪干细胞的培养和鉴定
目的体外分离培养大鼠ADSCs并进行鉴定,为PDE5-siRNA和iNOS基因修饰ADSCs移植治疗ED提供良好的种子细胞。
方法取SD大鼠腹股沟区皮下脂肪,采用Ⅰ型胶原酶消化法获取大鼠ADSCs,行传代培养,并取第4代细胞行CCK-8法测定细胞生长曲线。流式细胞术检测间充质干细胞免疫表型CD31、CD34、CD45、CD49d、 CD73、CD90、CD105和CD106表达情况初步鉴定ADSCs,再于成脂肪细胞、成平滑肌细胞、成血管内皮细胞和成神经细胞诱导培养基多向诱导分化不同时间后通过油红O染色、α-平滑肌肌动蛋白(a-SMA)和结蛋白(Desmin)、血管性血友病因子(vWF)、神经元核抗原(NeuN)和巢蛋白(Nestin)免疫荧光检测对诱导分化细胞进行鉴定。
结果第4代大鼠ADSCs生长曲线呈“S”形,为标准的细胞生长曲线。所培养的细胞阳性表达间充质干细胞相关阳性免疫表型CD49d、CD73、CD90、CD105和CD106,阳性率分别为(1.88±0.12)%、(4.05±0.13)%、(99.07±0.45)%、(4.68±0.23)%和(11.81±3.60)%,而造血干细胞表面分子CD34、CD45和血管内皮细胞表面分子CD31均为阴性表达,阳性率分别为(0.35±0.04)%、(0.38±0.01)%和(0.54±0.33)%。多向诱导分化鉴定显示,成脂肪诱导分化细胞油红O染色阳性,成平滑肌诱导分化细胞α-SMA和Desmin蛋白表达呈强阳性,成血管内皮诱导分化细胞vWF为阳性,成神经诱导分化细胞Nestin和NeuN表达阳性,对照组均为阴性,表明所培养的细胞具备多向诱导分化潜能。
结论成功从大鼠脂肪组织中分离培养ADSCs,为PDE5-siRNA和iNOS基因修饰干细胞提供了理想的细胞载体,从而为PDE5-siRNA和iNOS基因修饰ADSCs移植治疗ED提供良好的种子细胞。
第二部分大鼠脂肪干细胞的体外标记和示踪
目的比较研究氯甲基苯甲酰氨(CM-Dil)荧光活性染料和5-乙炔基-2’-脱氧尿嘧啶核苷(EdU)对大鼠ADSCs的体外标记示踪效果,为ADSCs移植治疗ED寻找最佳的干细胞示踪技术。
方法取第4代大鼠ADSCs,分别采用2μmol/L、4μmol/L、6μmol/L、8μmol/L、10μmol/L、12μmol/L、14μmol/L、16μmol/L、18μmol/L和20μmol/L CM-Dil不同标记浓度进行标记,通过流式细胞术和荧光显微镜筛选最佳标记浓度,并于最佳浓度标记后7d、14d、21d、28d和35d流式细胞术检测细胞阳性率及荧光强度。同时,50μmol/L EdU培养基分别标记ADSCs2h6^、12h、24h、48h、72h和96h,通过显色反应后于荧光显微镜下观察EdU标记不同时间的细胞阳性率,再于最佳标记时间标记后7d、14d、21d和28d检测EdU标记细胞阳性率。
结果CM-Dil标记浓度达10μmol/L时,ADSCs即发生饱和,标记效率即可近达100%,且该浓度对ADSCs的生长状态无明显影响。10μmol/L CM-Dil于标记后第7天和第14天细胞阳性率较高,组间差异无统计学意义(P>0.05)；第21天后荧光逐渐衰减,细胞阳性率开始降低(P<0.05),第28天明显降低但阳性率仍达80%以上。50μmol/L EdU标记ADSCs48h其标记效率达到峰值为最佳标记时间,EdU标记后第7天细胞阳性率开始降低(P<0.01),第14天急剧下降,第28天细胞阳性率仅为(14.39±2.67)%。
结论CM-Dil和EdU两者均可用于ADSCs标记、示踪,但CM-Dil标记具有操作简单、标记阳性率高、荧光持久等优点,适用于长期标记示踪,EdU仅适用于短期标记示踪。因此,CM-Dil标记示踪为ADSCs移植治疗ED的理想示踪技术。
第三部分
PDE5-siRNA和iNOS基因重组病毒载体的构建和鉴定
一、PDE5-siRNA重组慢病毒载体的构建
目的通过构建携带大鼠PDE5-siRNA的重组慢病毒载体(LV-PDE5-siRNA-GFP),为PDE5-siRNA植入ADSCs并稳定发挥干扰效应提供良好媒介。
方法直接选用课题组前期研究有效的PDE5-siRNA序列(GCAGCCGAATTCTT-TGATC),并合成含有靶序列的双链DNA Oligo。采用限制性内切酶Hpa I和Xho I对pFU-GW-RNAi慢病毒载体进行双酶切使其线性化,再通过T4DNA连接酶将双链DNA Oligo与线性化的pFU-GW-RNAi慢病毒载体连接并转化大肠杆菌DH5a感受态细胞,然后单克隆菌落采用PCR法鉴定,PCR鉴定阳性的克隆进一步行测序鉴定和比对分析,比对正确的克隆即为构建正确的目的载体。将pFU-GW-PDE5-siRNA重组慢病毒载体、pHelper1.0和pHelper2.0'慢病毒辅助包装质粒三质粒共转染293T细胞,转染48h后,收集293T细胞上清,然后进行病毒扩增、浓缩和纯化,最后采用孔稀释法测定慢病毒滴度,即病毒滴度(TU/mL)=最后一孔GFP阳性细胞数/病毒原液量×(1E+3)。
结果测序拼接序列中含有PDE5-siRNA序列(GCAGCCGAATTCTTTGATC)且匹配率为100%。1E+1μL、1E+OμL、1E-1μL、1E-2μL、1E-3μL、1E-4μL、1E-5μL1E-6μL和1E-7μL LV-PDE5-siRNA-GFP重组慢病毒稀释液转染293T细胞48h后,1E+1μL-1E-6μL转染孔均表达GFP,且GFP阳性细胞数随稀释倍数的增加而递减,1E-6μL转染孔仅检测到1个GFP阳性细胞,因此该病毒滴度为1E+9TU/mL。
结论携带大鼠PDE5-siRNA的重组慢病毒载体构建成功且获得较高病毒滴度,可用于ADSCs的基因修饰研究。
二、iNOS基因重组腺病毒载体的构建和鉴定
目的通过构建携带增强型绿色荧光蛋白(EGFP)和大鼠诱导型一氧化氮合酶(iNOS)基因的重组腺病毒载体,并观察由腺病毒介导的iNOS基因在293T细胞中的表达情况,为模拟iNOS基因在ADSCs中的表达提供可靠依据。
方法采用PCR方法钓取和扩增大鼠iNOS基因并获取iNOS基因全长片段。通过Age I限制性内切酶酶切pDC315-EGFP腺病毒载体使其线性化。通过In-Fusion交换酶将纯化的iNOS基因PCR产物与线性化的pDC315-EGFP腺病毒载体进行定向克隆连接,将连接产物转化感受态细胞,并采用PCR法鉴定单克隆菌落,对于PCR鉴定阳性的克隆进一步行测序鉴定和BLAT比对分析。将构建正确的pDC315-iNOS-EGFP质粒转染293T细胞,并于转染后48h采用Western Blot技术检测iNOS基因在293T细胞中的表达情况。成功构建的pDC315-iNOS-EGFP质粒,利用AdMax腺病毒包装系统包装产生重组腺病毒,病毒粗提液反复感染HEK293细胞行病毒扩增,病毒粗提液通过Adeno-X腺病毒纯化试剂盒进行纯化,最后采用倍比稀释法测定病毒滴度。取不同体积病毒原液转染293T细胞,于转染48h后通过Western Blot技术检测iNOS基因在蛋白水平的表达情况。
结果经PCR鉴定、限制性酶切分析、测序鉴定和目的质粒Western Blot表达检测鉴定,证实pDC315-iNOS-EGFP重组腺病毒载体构建成功。重组腺病毒包装成功,且病毒滴度为1.2E+10PFU/mL。iNOS基因重组腺病毒转染293T细胞48h后,Western Blot检测到约154KDa大小的条带,其与iNOS/GFP融合蛋白分子量大小基本相一致,表明腺病毒介导的iNOS基因成功在293T细胞中正确表达。
结论iNOS基因重组腺病毒载体构建成功且获得较高病毒滴度,为iNOS基因修饰ADSCs移植治疗ED奠定了良好的前期实验基础。
第四部分
PDE5-siRNA和iNOS基因在大鼠脂肪干细胞中的表达研究
目的观察PDE5-siRNA和iNOS基因在大鼠ADSCs中的表达效应,探讨PDE5-siRNA和iNOS基因修饰ADSCs移植治疗ED的可能性和有效性。
方法LV-PDE5-siRNA-GFP (MOI=70)和Ad-iNOS-EGFP (MOI=30)共转染第4代ADSCs,同时设立空白对照组(未转染组)、阴性对照组一(LV-NC-siRNA-GFP转染组)、阴性对照组二(Ad-NC-EGFP转染组)、阴性对照组三(LV-NC-siRNA-GFP+Ad-NC-EGFP共转染组)、阳性对照组-(LV-PDE5-siRNA-GFP转染组)和阳性对照组二(Ad-iNOS-EGFP转染组)。共转染后3d,采用Annexin V-PE/7-AAD凋亡试剂盒检测ADSCs凋亡情况,并于共转染第3天、5天、7天、10天和14天,分别采用实时荧光定量PCR和Western Blot技术检测PDE5和iNOS基因在mRNA水平和蛋白水平的表达情况。同时,分别采用硝酸还原酶法和ELISA法测定ADSCs培养上清中NO浓度和细胞内cGMP浓度。
结果实验组(LV-PDE5-siRNA-GFP+Ad-iNOS-EGFP）共转染组凋亡细胞比例为(3.04+0.58)%,与其他各组间差异无显著统计学意义(P>0.05)。实验组PDE5基因在mRNA水平和蛋白水平于转染后3d明显下调,5-7d PDE5-siRNA干扰效率达到峰值,10d后开始降低,但第14天干扰效率仍维持较高水平,与阳性对照组一差异无统计学意义(P>0.05)；腺病毒介导的iNOS基因于转染24h后在mRNA水平和蛋白水平出现显著上调,转染后5-7d达到峰值,10d后开始降低,14天后仍维持较高表达水平,其表达水平与阳性对照组二差异无统计学意义(P>0.05)；细胞内NO和cGMP浓度于转染3d后显著增加,5-7d达到峰值,于第10天出现明显降低后再次增加,至第14天仍维持较高浓度水平。
结论PDE5-siRNA和iNOS基因在ADSCs中有效、稳定共表达,且能同时有效提高细胞内NO和cGMP浓度水平,从而为PDE5-siRNA和iNOS基因修饰的ADSCs移植治疗ED的体内研究提供了有力的工具。
Objective To study the effects of rat PDE5gene specific small interference RNA (PDE5-siRNA) and iNOS single/double gene expression in rat adipose-derived stem cells (ADSCs), and investigate the feasibility and effectiveness of ADSCs modified with PDE5-siRNA and iNOS gene for erectile dysfunction (ED) therapy.
Methods Isolate and culture ADSCs from the adipose tissue of rat inguinal region by collagenase digestion. The cultured cells were identified not only by flow cytometer detection of mesenchymal stem cells (MSCs) related-immunophynotypes, but also by their capacities in the adipogenic, myogenic, endothelial and neurogenic differentiation. Then the fourth passage ADSCs were labeled with10μmol/L chloromethyl-benzamidodialkyl-carbocyanine (CM-Dil) and50μmol/L5-ethynyl-2'-deoxyuridine (EdU), and the labeling effects of CM-Dil and EdU on ADSCs were detected by flow cytometer and fluorescence microscope at7days,14days,21days and28days, respectively. The recombinant lentivirus vector expressing PDE5-siRNA (LV-PDE5-siRNA-GFP) and recombinant adenovirus vector carrying iNOS gene (Ad-iNOS-EGFP) were constructed, respectively. PDE5-siRNA and iNOS gene were cotransfected into ADSCs mediated by lentivirus and adenovirus. The experimental groups were designed as blank group, negative control group1(LV-NC-siRNA-GFP transfected group), negative control group2(Ad-NC-EGFP transfected group), negative control group3(LV-NC-siRNA-GFP and Ad-NC-EGFP cotransfected group), positive control group1(LV-PDE5-siRNA-GFP transfected group) and positive control group2(Ad-iNOS-EGFP transfected group) and experimental group (LV-PDE5-siRNA-GFP and Ad-iNOS-EGFP cotransfected group). The expression of PDE5and iNOS gene were examined with real-time fluorescence quantitative RT-PCR assay and western blot at3days,5days,7days,10days and14days, respectively. At the same time, the concentration of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) were assayed by nitrate reductase method and enzyme-linked immunosorbent assay (ELISA).
Results The cultured cells expressed CD49d, CD73, CD90, CD105and CD106, lacked CD31, CD34and CD45. Adipogenic, myogenic, endothelial and neurogenic differentiation cells can be stained with Oil Red O, a-SMA and Desmin, vWF, NeuN and Nestin, respectively, and the negative control was negative. The expression of PDE5gene was downregulated at3days, reached the peak at5to7days, and decreased at10days, and remained higher later. Compared to positive control group1(LV-PDE5-siRNA-GFP transfected group), no significant difference was noted (P>0.05). The mRNA and protein expression of iNOS gene mediated by recombinant adenovirus vector worked at24hours, reached a peak at5to7days, decreased at10days, and lasted at least2weeks. The expression level of PDE5was down-regulated by more than70percent from3to14days, significantly lower than negative control (P<0.001). However, there was no significant difference between cotransfected group and positive control group2(Ad-iNOS-EGFP transfected group), P>0.05. The NO concentration of cotransfected group and positive control group2was elevated at3days, reached the peak at5to7days, decreased at10days, and kept at least14days, but no greater than positive control group2(P>0.05). At the same time, the concentration of cGMP in cotransfected group and positive control groups increased at3days, reached the peak at5to7days, decreased at10days, but still kept at a high level later. The concentration level of cotransfected group was much higher than positive control groups (P<0.01), but no significant difference was noted between positive control group1and group2(P>0.05). The results showed that the concentration of NO and cGMP in ADSCs were elevated at3days, reached the peak at5to7days, decreased at10days but increased later, and lasted at least14days.
Conclusion PDE5-siRNA and iNOS gene mediated by lentivirus/adenovirus vector have successfully cotransfected and co-expressed in ADSCs. ADSCs modified by PDE5-siRNA and iNOS double gene could produce enough NO and more cGMP, which is more effective than single gene modified. Therefore, CM-Dil labeling ADSCs modified with PDE5-siRNA and iNOS genes could be the ideal seed cells in the future treatment of ED.
PartⅠ
Culture and Identification of Rat Adipose-Derived Stem Cells
Objective To isolate and culture rat ADSCs in vitro, and provide the available seed cells for PDE5-siRNA and iNOS gene modified stem cells in the future treatment of ED.
Methods Obtain adipose tissue from rat inguinal region by surgical resection, and isolate and culture ADSCs by collagenase type I digestion method. The fourth generation cell growth curve was described with cell counting kit-8(CCK-8). Mesenchymal stem cells (MSCs) related-immunophynotypes CD31, CD34, CD45, CD49d, CD73, CD90, CD105and CD106were detected by flow cytometer detection. The characterization of ADSCs was also arrayed by inducing in the adipogenic, myogenic, endothelial and neurogenic differentiation, and identified with Oil Red O staining, a-SMA and Desmin staining,vWF staining, NeuN and Nestin staining, respectively.
Results The growth curve of the fourth passage cultured cells was presented typical "S" shape. The cultured cells expressed CD49d, CD73, CD90, CD105and CD106, lacked CD31, CD34and CD45. The positive rate was (1.88±0.12)%,(4.05±0.13)%,(99.07±0.45)%,(4.68±0.23)%,(11.81±3.60)%,(0.35±0.04)%,(0.38±0.01)%and (0.54±0.33)%, respectively. Adipogenic, myogenic, endothelial and neurogenic differentiation cells can be stained with Oil Red O, a-SMA and Desmin, vWF, NeuN and Nestin, respectively. However, the negative control was negative.
Conclusion ADSCs have been successfully isolated and cultured from rat adipose tissue, and could be used as ideal cell carrier for PDE5-siRNA and iNOS gene modified stem cells, provide the available seed cells for PDE5-siRNA and iNOS gene modified stem cells in the future treatment of ED.
Part Ⅱ
Cell Tracker Labeling for Rat Adipose-Derived Stem Cells
Objective To compare the labeling effects of CM-Dil and EdU on rat ADSCs, and seek the optimal stem cell labeling technique applied to ED therapy.
Methods The fourth generation ADSCs were labeled with2μmol/L,4μmol/L,6μmol/L,8μmol/L,10μmol/L,12μmol/L,14μmol/L,16μmol/L,18μmol/L and20μmol/L CM-Dil solution. The optimal labeling concentration was screened by flow cytometer and fluorescence microscope. Then the CM-Dil positive cell rate was detected by flow cytometer and fluorescence microscope at7days,14days,21days and28days, respectively. In order to seek the optimal labeling time of EdU for ADSCs, ADSCs were labeled with50μmol/L EdU containing medium incubated for2h,6h,12h,24h,48h,72h and96h. EdU positive cell rate was detected by fluorescence microscope at7days,14days,21days and28days, respectively.
Results The optimal labeling concentration of CM-Dil for ADSCs was10μmol/L, and the positive cell rate was near to100%.10μmol/L CM-Dil had no bad effects on the growth of ADSCs. The positive cell rate was higher at7days and14days (P>0.05), decreased at21days (P<0.05), but still kept more than80%at28days. The labeling efficiency of EdU reached the peak incubating for48hours, and decreased at72hours. The positive rate of EdU labeling cells was (87.1±1.8)%, and decreased at7days (P<0.01), and the positive rate was only (14.4±2.7)%at28days.
Conclusion CM-Dil and EdU are suitable for ADSCs tracker labeling. However, CM-Dil gains much more advantages than EdU in long-term tracing, which may be an ideal and optimal ADSCs tracing technology in the future treatment of ED.
Part Ⅲ
Construction and Identification of Recombinant Viral Vector Carrying Rat PDE5-siRNA and iNOS Gene
PartⅢ-A
Construction of Recombinant Lentivirus Vector Expressing RatPDE5-siRNA
Objective Construct recombinant lentivirus vector expressing rat PDE5-siRNA (LV-PDE5-siRNA-GFP), and provide a good transfer media for PDE5-siRNA expression in ADSCs thereby.
Methods The effective interfere sequence for PDE5(GCAGCCGAATTCTTTGATC) was obtained from our prophase research results. Double-stranded DNA Oligo containing PDE5-siRNA sequence was synthesized and connected with linearized pFU-GW-RNAi recombinant lentivirus vector by T4DNA ligase. There recombinant pFU-GW-PDE5-siRNA recombinant lentivirus vector was transformed into competent cells. The positive recombination was selected by polymerase chain reaction (PCR) and further identified by sequencing analysis.293T cells were infected by successfully constructed pFU-GW-PDE5-siRNA lentivirus vector together with pHelper1.0and pHelper2.0plasmid. Cellular supernatant of transfected293T cells was harvested, concentrated and purified at48hours. The lentivirus titer was determined by dilution method.
Results The sequencing results showed that targeted PDE5-siRNA sequence GCAGCC-GAATTCTTTGATC was was consistent with pFU-GW-PDE5-siRNA lentivirus vector sequence. The lentivirus titer was1E+9TU/mL.
Conclusion Recombinant lentivirus vector carrying rat PDE5-siRNA was successfully constructed and high lentivirus titer was obtained, which could be applied to further study.
PartⅢ-B
Construction and Identification of Recombinant Adenovirus Vector Harboring Rat iNOS Gene
Objective Construct recombinant adenovirus over-expression vector harboring enhanced green fluorescent protein (EGFP) and rat iNOS gene, and observe iNOS gene expression mediated by recombinant adenovirus in293T cells, which may provide reliable basis for the possibility of iNOS gene expression in ADSCs.
Method The full-length iNOS gene was fished and amplified by PCR. pDC315-EGFP adenovirus vector was linearized with Age I digestion, then connected with iNOS gene. There recombinant pDC315-iNOS-EGFP adenovirus vector was transformed into competent cells and identified by restriction enzyme digestion, PCR and DNA sequencing.293T cells were transfected with confirmed pDC315-iNOS-EGFP plasmid. The expression of pDC315-iNOS-EGFP plasmid was detected by western blot assay. The recombinant adenovirus was generated by AdMax packaging system in HEK293cells. Raw adenovirus was harvested, concentrated and purified by Adeno-X Maxi purification kit. The lentivirus titer was determined by doubling dilution method. The iNOS gene recombinant adenovirus was identified by western blot in293T cells.
Results pDC315-iNOS-EGFP adenovirus vector was successfully constructed and confirmed by restriction enzyme digestion, PCR, DNA sequencing and western blot assay. The adenovirus titer was1.2E+10PFU/mL. The iNOS/GFP fusion proteins were successfully expressed in293T cells observing by western blot at48hours.
Conclusion Recombinant adenovirus over-expression vector harboring rat iNOS gene was successfully constructed and expressed in293T cells, which could be further applied to the modification of ADSCs.
Part IV
The Expression of PDE5-siRNA and iNOS Gene in Rat Adipose-Derived Stem Cells
Objective To compare the effects of rat PDE5-siRNA and iNOS single/double gene rat on ADSCs, and investigate the possibility and feasibility of ADSCs modified with PDE5-siRNAand iNOS gene for future ED therapy by grafting into corpus cavernosum.
Methods The experiment groups were designed as blank group, negative control group1(LV-NC-siRNA-GFP transfected group), negative control group2(Ad-NC-EGFP transfected group), negative control group3(LV-NC-siRNA-GFP and Ad-NC-EGFP cotransfected group), positive control group1(LV-PDE5-siRNA-GFP transfected group) and positive control group2(Ad-iNOS-EGFP transfected group) and experimental group (LV-PDE5-siRNA-GFP and Ad-iNOS-EGFP cotransfected group). ADSCs were cotransfected with LV-PDE5-siRNA-GFP (MOI=70) and Ad-iNOS-EGFP (MOI=30). The apoptosis of transfected ADSCs was detected by Annexin V-PE/7-AAD apoptosis kit and flow cytometry at3days. The expressions of PDE5and iNOS gene in ADSCs were detected by real time RT-PCR and western blot assay at3days,5days,7days,10days and14days. Nitric oxide and cGMP were determined by Nitrate/Nitrite Assay Kit cultured with lOmM L-arginine containing medium for24hours and ELISA Kit cultured with10μM sodium nitroprusside containing medium for2minutes, respectively.
Results The percentage of cotransfected ADSCs apoptosis was (3.04±0.58)%, and no significant difference was noted between the7groups (P>0.05). PDE5-siRNA worked at3days, reached a peak at5to7days, decreased at10days, and lasted at least14days. The expression level of PDE5was down-regulated by more than70percent from3to14days, significantly lower than negative control (P<0.001). iNOS gene began to express at24hours, reached a peak at4to5days, and decreased at10days. There was an overexpression of iNOS gene in ADSCs modified with iNOS (P<0.001). The level of NO was greatly higher than negative control (P<0.01), but no greater than iNOS single gene modified control (P>0.05). The concentration of cGMP was significantly greater than PDE5-siRNA/iNOS single gene modified control (P<0.01). The activation effects of PDE5-siRNA and iNOS double gene on NO/cGMP in ADSCs were kept at least2weeks.
Conclusion PDE5-siRNA and iNOS gene were coexpressed in ADSCs, and produced enough NO and more cGMP thereby. Therefore, ADSCs modified with PDE5-siRNA and iNOS genes could be the ideal seed cells in the future treatment of ED by grafting into corpus cavernosum.
引文
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1. Albersen M, Orabi H, Lue TF. Evaluation and treatment of erectile dysfunction in the aging male:a mini-review. Gerontology,2012,58(1):3-14.
2. Gratzke C, Angulo J, Chitaley K, et al. Anatomy, physiology, and pathophysiology of erectile dysfunction. J Sex Med,2010,7(1 Pt 2):445-475.
3. O'Connor DM, O'Brien T. Nitric oxide synthase gene therapy:progress and prospects. Expert Opin Biol Ther,2009,9(7):867-878.
4. Huang YC, Ning H, Shindel AW, et al. The Effect of Intracavernous Injection of Adipose Tissue-Derived Stem Cells on Hyperlipidemia-Associated Erectile Dysfunction in a Rat Model. J Sex Med,2010,7(4 Pt 1):1391-1400.
5. Garcia MM, Fandel TM, Lin G, et al. Treatment of erectile dysfunction in the obese type 2 diabetic ZDF rat with adipose tissue-derived stem cells. J Sex Med,2010,7(1 Pt 1):89-98.
6. Bunnell BA, Flaat M, Gagliardi C, et al. Adipose-derived stem cells:isolation, expansion and differentiation. Methods,2008,45(2):115-120.
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14. Albersen M, Fandel TM, Lin G, et al. Injections of adipose tissue-derived stem cells and stem cell lysate improve recovery of erectile function in a rat model of cavernous nerve injury. J Sex Med,2010,7(10):3331-3340.
15. Qiu X, Villalta J, Ferretti L, et al. Effects of Intravenous injection of adipose-derived stem cells in a rat model of radiation therapy-induced erectile dysfunction. J Sex Med, 2012,9(7):1834-1841.
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1. Sun C, Lin H, Yu W, et al. Neurotrophic effect of bone marrow mesenchymal stem cells for erectile dysfunction in diabetic rats. Int J Androl,2012,35(4):601-607.
2. Garcia MM, Fandel TM, Lin G, et al. Treatment of erectile dysfunction in the obese type 2 diabetic ZDF rat with adipose tissue-derived stem cells. J Sex Med,2010,7 (1 Pt 1):89-98.
3. Huang YC, Ning H, Shindel AW, et al. The Effect of Intracavernous Injection of Adipose Tissue-Derived Stem Cells on Hyperlipidemia-Associated Erectile Dysfunction in a Rat Model. J Sex Med,2010,7(4 Pt 1):1391-1400.
4. Albersen M, Fandel TM, Lin G, et al. Injections of adipose tissue-derived stem cells and stem cell lysate improve recovery of erectile function in a rat model of cavernous nerve injury. J Sex Med,2010,7(10):3331-3340.
5. Bunnell BA, Flaat M, Gagliardi C, et al. Adipose-derived stem cells:isolation, expansion and differentiation. Methods,2008,45(2):115-120.
6. Weir C, Morel-Kopp MC, Gill A, et al. Mesenchymal stem cells:isolation, characterisation and in vivo fluorescent dye tracking. Heart Lung Circ,2008,17(5): 395-403.
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11.李建伟,胡蕴玉,魏义勇,等.5-溴脱氧尿嘧啶核苷标记示踪脂肪成体干细胞体 内成骨分化的研究.中华实验外科杂志,2008,25(6)：697-698.
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14. Albersen M, Orabi H, Lue TF. Evaluation and treatment of erectile dysfunction in the aging male: a mini-review. Gerontology,2012,58(1):3-14.
1. Burnett AL. Erectile dysfunction management for the future. J Androl,2009,30(4): 391-396.
2. Shamloul R, Ghanem H. Erectile dysfunction. Lancet,2013,381(9861):153-165.
3. Kendirci M, Teloken PE, Champion HC, et al. Eur Urol,2006,50(6):1208-1222.
4. Yoshimura N, Kato R, Chancellor MB, et al. Gene therapy as future treatment of erectile dysfunction. Expert Opin Biol Ther,2010,10(9):1305-1314.
5. O'Connor DM, O'Brien T. Nitric oxide synthase gene therapy: progress and prospects. Expert Opin Biol Ther,2009,9(7):867-878.
6. Qiu X, Sun C, Yu W, et al. Combined strategy of mesenchymal stem cell injection with vascular endothelial growth factor gene therapy for the treatment of diabetes-associated erectile dysfunction. J Androl,2012,33(1):37-44.
7. Gou X, He WY, Xiao MZ, et al. Transplantation of endothelial progenitor cells transfected with VEGF165 to restore erectile function in diabetic rats. Asian J Androl, 2011,13(2):332-338.
8. Bochinski D, Lin GT, Nunes L, et al. The effect of neural embryonic stem cell therapy in a rat model of cavernosal nerve injury. BJU Int,2004,94(6):904-909.
9. Bivalacqua TJ, Deng W, Kendirci M, et al. Mesenchymal stem cells alone or ex vivo gene modified with endothelial nitric oxide synthase reverse age-associated erectile dysfunction. Am JPhysiol Heart Circ Physiol,2007,292(3):H1278-1290.
10. Harraz A, Shindel AW, Lue TF. Emerging gene and stem cell therapies for the treatment of erectile dysfunction. Nat Rev Urol,2010,7(3):143-152.
11.李忠远,刘继红,封江南,等.腺病毒载体介导shRNA抑制大鼠阴茎海绵体平滑肌细胞PDE5基因表达的研究.中华泌尿外科杂志,2008,29(2)：131-132.
12.潘运高,刘继红,詹鹰,等.腺病毒载体介导PDE5-shRNAs对大鼠阴茎海绵体平滑肌细胞游离钙的影响.中华男科学杂志,2009,15(10)：882-885.
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