Gene transfer to human trabecular meshwork cells in vitro and ex vivo using HIV-based lentivirus

AIM: To investigate whether the enhanced green fluorescent protein(EGFP) reporter gene could be transferred into human trabecular meshwork(HTM) cells by a HIV-based lentivirus both in vitro and ex vivo.METHODS: The HIV-based lentivirus that contains an EF1-α promoter driving EGFP expression cassette was constructed following the standard molecular cloning methods. The cultured HTM cells were transduced at a range of multiplicity of infection(MOI) with HIV-based lentivirus. EGFP positive cell populations were detected by flow cytometry. Human anterior eye segments were cultured with perfusion culture system and transfected by HIV-based lentivirus with a 1 ×108transducing unit(TU) virus in perfusion liquid. The intraocular pressure was recorded every 8h for 21 d. The expression of EGFP in the anterior segment of the human eye was detected by fluorescence microscopy. Furthermore, the distribution of EGFP expression was confirmed by anti-EGFP immunohistochemical staining.RESULTS: The HIV-based lentivirus which contains an EF1-α promoter driving EGFP expression cassette was constructed successfully. After HTM cells were transduced with HIV-based lentivirus containing EGFP in vitro, the ratio of EGFP positive cells to the total cell number reached 92.3%, with the MOI of 15. After the lentivirus containing EGFP were used to transduce human anterior eye segments, the EGFP could be directly detected by fluorescence microscopy in vivo.Immunohistochemistry staining revealed that 88.19%EGFP-positive trabecular meshwork(TM) cells were observed in the human anterior segment. Nevertheless,the intraocular pressure in the lentivirus-transduced group kept constant when compared with control group(P 】0.05).CONCLUSION: EGFP gene could be efficiently transferred into HTM cells both in vitro and ex vivo by using HIV-based lentivirus.

Lentivirus介导RNAi抑制人胶质瘤干细胞STAT3基因生物学效应观察

目的了解慢病毒载体对胶质瘤干细胞的亲嗜性及对靶基因表达的干扰效率,初步探索干扰STAT3基因对胶质瘤干细胞增殖的影响。方法构建STAT3基因shRNA的慢病毒表达载体,经293T细胞包装后,获得可表达STAT3基因shRNA的慢病毒颗粒;活性载体病毒感染人原代胶质瘤干细胞后流式细胞分析细胞感染效率;实时定量多聚酶链反应(PCR)和Western blot检测细胞STAT3基因mRNA和蛋白表达及活化水平;增殖分析试剂盒测定细胞生长曲线,流式细胞分析细胞周期分布。结果在病毒感染比率值20:1时慢病毒载体对人原代胶质瘤干细胞的感染效率为98.6%;细胞感染可表达STAT3基因shRNA的载体慢病毒后,STAT3基因mRNA显著下降,抑制率为84.3%,STAT3蛋白表达下降81.5%,活化的pSTAT3下降97.9%;胶质瘤干细胞STAT3表达、活化受抑后细胞生长显著变慢,G1期细胞比例显著增高。结论慢病毒载体对人原代胶质瘤干细胞有着很高的感染效率,介导的RNAi可显著抑制靶基因的表达与活化,是对人胶质瘤干细胞基因功能研究的理想工具。人胶质瘤干细胞STAT3基因受抑后细胞生长显著变慢,出现G1期阻滞。

Cathepsin B-RNAi-lentivirus新生血管形成的研究抑制小鼠视网膜

背景 视网膜新生血管性疾病是一种严重影响视力的眼病.研究表明,cathepsin B参与新生血管的生成,寻找抑制视网膜新生血管形成的药物可为此类疾病的分子机制研究提供依据. 目的 研究cathepsin B-RNAi-lentivirus对小鼠视网膜新生血管的抑制作用. 方法 将7日龄清洁级C57BL/6J小鼠与其母鼠共同置于氧体积分数为（75±2）％的密闭氧箱内5d,制作视网膜新生血管模型,5d后返回正常环境.应用随机数字表法将60只小鼠随机分为模型对照组、阴性对照组和基因治疗组,每组20只.模型对照组小鼠不给予任何药物干预,阴性对照组小鼠玻璃体腔内注射空载体与辅助载体包装成的无目的基因的空病毒颗粒（NC-GFP-LV）1 μl,基因治疗组以同样的方法注射eathepsin B-RNAi-lentivirus 1μl.注射5d后各组分别应用随机数字表法随机处死7只小鼠,获取14只眼的视网膜,采用real-time PCR法检测小鼠视网膜中cathepsin BmRNA的表达量（2△△Ct）,另外同时间点同法分别处死8只小鼠,获取16只眼球的视网膜,采用Western blot法检测各组小鼠视网膜中cathepsin B蛋白的相对表达量（cathepsin B/β-actin）.注射5d后各组分别取剩余5只小鼠行异硫氰酸葡聚醛荧光素（FITC-dextran）心脏灌注,并处死动物,制备10只眼的视网膜铺片,荧光显微镜下观察和比较各组小鼠视网膜新生血管的形态和数量. 结果 基因治疗组小鼠视网膜中cathepsin B mRNA的表达水平（2-△△Ct）为0.740.12,明显低于模型对照组及阴性对照组的1.66±0.17和1.58±0.29,差异均有统计学意义（q=0.746、1.588,P＜0.01）.基因治疗组小鼠视网膜中cathepsin B蛋白的表达水平（cathepsin B/β-actin）为0.64±0.06,明显低于模型对照组及阴性对照组的0.93±0.09和0.96±0.09,差异均有统计学意义（q=0.637、0.894,P＜0.01）.小鼠视网膜荧光染色结果显示,模型对照组和阴性对照组小鼠视网膜新生血管分层及分支多,血管走形扭曲,部分血管可见荧光素渗漏；基因治疗组小鼠血管走形较直,血管分支少,新生血管数量少. 结论 Cathepsin B-RNAi-lentivirus转染能有效抑制小鼠视网膜新生血管的形成.

Lentivirus介导表达多基因的人胚基因工程神经干细胞的实验研究

目的:探索以Lentivirus为载体,构建同时携带并表达多基因的基因工程人胚神经干细胞(hum an neu鄄ral stem cell,hNSC)的可行性,为脊髓损伤治疗的研究提供材料。方法:培养和鉴定hNSC;用携带绿色荧光蛋白(green fluorescence protein,GFP)和神经营养因子-3(neurotrophic factor-3,NT-3)的Lentivirus转染hNSC;用荧光显微镜观察、鼠胚背根神经结培养(dorsal root ganglion,DRG)和Slot blot等方法检测基因工程hNSC的多基因表达情况。结果:培养获得了大量的hNSC;荧光显微镜观察到几乎100%的hNSC表达GFP;基因工程hNSC的培养液能促使大鼠DRG旺盛生长;Slot blot检测到基因工程hNSC能高效分泌NT-3蛋白。结论:以Lentivirus为载体能构建同时携带并稳定表达多基因的基因工程hNSC,为脊髓损伤治疗的基础研究及进一步临床应用提供了有价值的细胞资源。

Lentivirus载体对许旺细胞的转染效率

背景:近年来研究较多的LV载体,因其强大的转染能力以及较高的转染效率等特点,在基因转染的实验中应用越来越广泛。目的:进一步验证Lentivirus载体在体外对原代许旺细胞的转染效率。方法:以Lentivirus三质粒系统构建病毒载体,在体外分别以MOI值为1,5,10,20,40对原代大鼠许旺细胞进行转染,转染后第1,3,5,7,9天在荧光显微镜下观察Lentivirus携带的荧光表达情况,并在显微镜的计数方格内计算细胞的转染情况。发出绿色荧光的为转染成功的许旺细胞,否则认为没有转染病毒载体的,从而算出转染效率。结果与结论:在病毒转染3 d后,在各不同MOI值的培养孔中,均能观察到极少量的荧光反应,在第5天时,荧光数量较前明显增多。第7天时达到高峰,第9天的荧光数量与第7天变化不明显。从细胞转染效率来看,不同的MOI值明显不同,MOI值为1时的转染效率约为45%,MOI值为5时为80%,MOI值为10时为90%,MOI值为20时为78%,MOI值为40时转染效率为70%。

Inhibition of choroidal neovascularization by lentivirusmediated PEDF gene transfer in rats

AIM: To evaluate the effects of lentivirus-mediated pigment epithelium-derived factor (PEDF) gene transfer performed in treatment of rats with established choroidal neovascularization (CNV), and investigates the mechanism by which PEDF inhibits CNV in rats. METHODS: Brown Norway (BN) rats (n=204) were induced by exposure to a laser, and then randomly assigned to 3 groups: no treatment; treatments with intravitreal injection of lentivirus-PEDF-green fluorescent protein (GFP) or lentivirus-control GFP (free fluorescent protein). Following induction and treatment, the CNV tissue was assessed for form, size and vessel leakage by fluorescein fundus angiography (FFA), optical coherence tomography (OCT), histopathology, and examination of choroidal flat mounts. VEGF, Flk-1, and PEDF expression were evaluated by real-time polymerase chain reaction (PCR) and Western blot. RESULTS: A stable laser-induced rat model of CNV was successfully established, and used to demonstrate lentivirus-mediated REDO gene transfer by intravitreal injection. Expression of green fluorescence labelled PEDF was observed in the retina up to 28d after injection. An intravitreal injection of lentivirus-PEDF-GFP at 7d led to a significant reduction in the size, thickness and area of CNV showed by FFA, OCT and choroidal flat mounts. PEDF was up-regulated while VEGF and Flk-1 were down-regulated in the lentivirus-PEDF-GFP group. The differences in VEGF and Flk-1 expression in the control and lentivirus-PEDF groups at 7, 14, 21 and 28d after laser induction were all statistically significant. CONCLUSION: Lentivirus-mediated PEDF gene transfer is effective for use in treatment of laser-induced CNV, and PEDF exerts its therapeutic effects by inhibiting expression of VEGF and Flk-1.

Lentivirus carrying the Atoh1 gene infects normal rat cochlea

Lentivirus carrying the Atohl gene can infect Corti＇s organ and express a hair-like cell surface marker in the supporting cell area. However, expression of the gene carried by adenovirus is instantaneous, which undoubtedly limits its clinical application. Lentivirus acts as a carrier that can stably and continuously express genes. In this study, the cochlear structure and hearing level were not affected, and Atohl gene carried by lentivirus promoted the production of hair-like cells in the cochlear supporting cell area. This led to expression of the hair-like cell surface marker myosin 7a 30 days after lentivirus carrying Atohl was microinjected into the cochlear round window of rats.

Construction of lentivirus vectors carrying alphastatin gene and its secretion expression in human umbilical vein endothelia cells

Objective To construct lentivirus vectors carrying alphastatin gene,test its secretion expression in human umbilical vein endothelia cells(HUVECs)and observe its effects on growth,migration and tube formation of HUVECs.Methods We constructed recombinant lentivirus vectors of NT4-alphastatin fusion gene containing neurotrophin-4 signal peptide,pro-region sequences and alphastatin,then transfected the recombinant lentivirus vectors into HUVECs to obtain secretory protein alphastatin and test its anti-angiogenic activities in vitro.Results Our data showed that recombinant self-inactivating lentivirus vectors of NT4-alphastatin were successfully constructed,and stable NT4-alphastatin transduced HUVECs were capable of sustainably secreting alphastatin which significantly suppressed HUVECs migration and differentiation but not VEGF-induced proliferation.Conclusion This report represents the first time on the use of lentivirus-based vectors to deliver alphastatin,the endogenous angiogenesis inhibitor,and reveals the potential utility of anti-angiogenic gene therapy with lentivirus vectors for treating cancer.

Lentivirus-mediated RNA Interference and Over-expression of CDK2AP1 CDNA Regulate CDK2AP1 Expression in Human Lung Cancer A549 Cells

Cyclin-dependent kinase 2-associated protein 1（CDK2AP1）, a cell growth inhibitory factor, is abnormally expressed in cancer cells, and might be implicated in the development of lung cancer. However, no studies on the function of CDK2AP1 in human lung cancer have been yet reported. In this study, overexpressing lentiviral vectors containing full-length CDK2AP1 cDNA and CDK2AP1 shRNA（short hairpin RNA） were constructed. Our results show that infecting A549 cells with lentivirus containing CDK2AP1 shRNA or full-length CDK2AP1 cDNA results in significantly down- or up-regulated the expression of CDK2AP1, respectively, thereby providing reliable tools for studying the function of CDK2AP1 in pulmonary carcinogenesis. Our data of MTT assay and flowcytometric analysis demonstrate that CDK2AP1 plays an important role in the proliferation/growth and cell cycling of A549 cells in vitro, and further investigation into its underlying mechanism of pulmonary carcinogenesis is needed.

Construction of a recombinant lentivirus containing human microRNA-7-3 and its inhibitory effects on glioma proliferation

In the present study, we constructed a lentivirus, FIV-CMV-GFP-miR-7-3, containing the microRNA-7-3 gene and the green fluorescent protein gene, and used it to transfect human glioma U251 cells. Fluorescence microscopy showed that 80% of U251 cells expressed green fluorescence. Real-time reverse transcription PCR showed that microRNA-7-3 RNA expression in U251 cells was significantly increased. Proliferation was slowed in transfected U251 cells, and most cells were in the G1 phase of the cell cycle. In addition, the expression of the serine/threonine protein kinase 2 was decreased. Results suggested that transfection with a lentivirus carrying microRNA-7-3 can effectively suppress epidermal growth factor receptor pathway activity in U251 cells, arrest cell cycle transition from GI phase to S phase and inhibit glioma cell growth.

Lentivirus-mediated Persephin overexpression in Parkinson's disease rats

Persephin, together with glial cell line-derived neurotrophic factor and neurturin, has a neurotrophic effect and promotes the survival of motor neurons cultured in vitro. In this study, dopaminergic neurons in the substantia nigra of rats were transfected with the Persephin gene. One week later 6-hydroxydopamine was injected into the anterior medial bundle to establish a Parkinson＇s disease model in the rats. Results found that the number of dopaminergic neurons in the substantia nigra increased, tyrosine hydroxylase expression was upregulated and concentrations of dopamine and its metabolites in corpus striatum were increased after pretreatment with Persephin gene. In addition, the rotating effect of the induced Parkinson＇s disease rats was much less in the group pretreated with the Persephin gene. Persephin has a neuroprotective effect on the 6-hydroxydopamine-induced Parkinson＇s disease through protecting dopaminergic neurons.

Diversity of Primate Lentiviruses Rebooted

Highlight: The present report reveals for the first time natural lentiviral infection of wild Indian NHPs, rhesus monkeys (Macaca mulatta) and langurs (Semnopithecus entellus) by SIVs that are phylogenetically diverse from all known SIVs, including “SIVmac”, which infects captive rhesus monkeys. The novel SIVs are intriguingly homologous to HIV-1, based on serology and partial lentiviral genomic sequence analyses. Diverse lenti-viruses infect human and nonhuman primates (NHPs). There are more than 45 different “species-specific” simian immunodeficiency viruses (SIVs) that infect their cognate NHP hosts in natural habitats in Africa. Indian NHPs are not known to be infected by SIVs in the wild. Conventionally SIVs are named after their natural hosts, except for SIVmac, which infects captive rather than wild rhesus macaques. SIVmac is therefore a misnomer. It is a genetic variant of the African SIVsmm, which infects wild African sooty mangabey monkeys. SIVsmm is the progenitor of human immunodeficiency virus (HIV-2), while SIVcpz that infects wild chimpanzees is the progenitor of HIV-1. Although natural infections cannot be easily studied in wild NHP populations, we have previously reported co-infection of wild Indian NHPs by other retroviruses: simian retroviruses (SRVs) and Simian Foamy viruses (SFV). Apart from zoonosis, transmission of pathogens from humans to animals: anthroponosis, has also been reported in literature.

Lentivirus-mediated SMO RNA interference inhibits SMO expression and cell proliferation,and affects the cell cycle in LNCaP and PC3 cancer cell lines

Smoothened （SMO） is an important member of the Hedgehog signaling pathway. We constructed a specific recombinant lentiviral vector for RNA interference,targeting the SMO gene （NM_005631） to observe its effect on SMO expression,cell proliferation and the cell cycle in the human androgen-sensitive prostate cancer cell line,LNCaP,and in the androgen-independent prostate cancer cell line,PC3. Four siRNA sequences were designed and inserted into a lentiviral vector pGCSIL-GFP to construct four recombinant vectors. The vector with the highest interfering efficiency was co-transfected with packaging vectors （pHelper1.0 and pHelper2.0） in 293T cells to assemble lentivirus particles by liposome for infecting LNCaP and PC3 cell lines,respectively. The expression level of SMO mRNA,tumor cell proliferation and cell cycle were measured by quantitative realtime polymerase chain reaction （qRT-PCR）,3-（4,5）-dimethylthiahiazo （-z-yl）-3,5-di-phenytetrazoliumromide （MTT） assay and flow eytometry,respectively. Sequence results showed that recombinant lentiviral vectors were constructed successfully.pGCSIL-GFP-723 had the highest interfering efficiency,named Lv-SIL-SMO723 after co-transfection,with which LNCaP and PC3 cell lines were infected. Compared with the control groups,results showed significantly decreased （P〈0.05） SMO mRNA expressions of LNCaP and PC3,lower mean percentage of S-phase cells and higher mean percentage of G_2/M phase cells,as well as obviously slow proliferation （P〈0.01） of LNCaP in the infected group. Yet,the proliferation of PC3 was not altered （P〉0.05）. In conclusion,the recombinant lentivirus particles were able to suppress SMO expression,regulate the cell cycle in the LNCaP and PC3 cell lines and markedly inhibit proliferation of LNCaP cells but not PC3 cells.

Lentivirus-mediated shRNA interference targeting STAT3 inhibits human pancreatic cancer cell invasion

AIM: To investigate RNA interference targeting signal transducer and activator of transcription-3 (STAT3) on invasion of human pancreatic cancer cells. METHODS: We constructed three plasmids of RNA interference targeting the STAT3 gene. After LV (lentivirus)-STAT3siRNA (STAT3 small interfering RNA) the vector was transfected into the human pancreatic cell line, SW1990 and cell proliferation was measured by the MTT assay. Flow cytometry was used to assess cell cycle. Vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) mRNA and protein expression were examined by quantitative PCR and western blotting, respectively. The invasion ability of SW1990 cells was determined by cell invasion assay. RESULTS: We successfully constructed the LVSTAT3siRNA lentivirus vector and proved that it can suppress expression of STAT3 gene in SW1990 cells. RNA interference of STAT3 by the LV-STAT3siRNA construct signifi cantly inhibited the growth of SW1990 cells, in addition to signifi cantly decreasing both VEGF and MMP-2 mRNA and protein expression. Moreover, suppression of STAT3 by LV-STAT3siRNA decreased the invasion ability of SW1990 cells.CONCLUSION: The STAT3 signaling pathway may provide a novel therapeutic target for the treatment of pancreatic cancer since it inhibits the invasion ability of pancreatic cancer cells.

Lentivirus Mediated Gene Manipulation in Trophectoderm of Porcine Embryos

Development of tools that can manipulate gene expression specifically and efficiently in the trophectoderm（TE） lineage would greatly aid understanding the roles of different genetic pathways in TE versus embryonic lineages. Here, we showed first time that short-term lentivirus infection of porcine blastocysts could lead to rapid expression of transgene specifically in TE cells. Efficient TE-specific gene knockdown could also be achieved by lentivirus-mediated pol III-driven short hairpin RNA（shRNA） and TE-specific gene expression could be temporal controlled efficiently by combining this system with Tet-On system. This lentivirus lineage-specific infection system would facilitate gene function studies in porcine pre-implatation embryos by specifically knockdown or overexpression of these genes in TE.

GENETIC ENGINEERING NEURAL STEM CELL MODIFIED BY LENTIVIRUS FOR REPAIR OF SPINAL CORD INJURY IN RATS

Objective To explore the feasibility for therapy of spinal cord injury (SCI) by genetic engineering neural stem cell (NSC) modified by lentiviral vector. Methods Following the construction of the genetic engineering NSC modified by lentivirus to secrete both neurotrophic factor-3 (NT-3) and green fluorescence protein (GFP), hemisection of spinal cord at the level of T_ 10 was performed in 56 adult Wistar rats that were randomly divided into 4 groups (n=14), namely 3 therapeutic groups and 1 control group. The therapeutic groups were dealed with NSC, genetic engineering NSC, and concentrated lentiviral supernatant which carries both GFP and NT-3, respectively. Then used fluorescence microscope to detect the transgenic expression in vitro and in vivo, migration of the grafted cells in vivo, and used the Basso, Beattie, and Bresnahan (BBB) open-field locomotor test to assess the recovery of function. Results The transplanted cells could survive for long time in vivo and migrate for long distance. The stable transgenic expression could be detected in vivo. The hindlimb function of the injured rats in 3 therapeutic groups, especially those dealed with genetic engineering NSC, improved obviously. Conclusion It is feasible to combine NSC with lentivirus for the repair of SCI. NSC modified by lentivirus to deliver NT-3, acting as a source of neurotrophic factors and function cell in vivo, has the potential to participate in spinal cord repair.

Lentivirus mediated shRNA interference targeting MAT2B induces growth-inhibition and apoptosis in hepatocelluar carcinoma

AIM: To investigate the effects of lentivirus vector mediated short hairpin RNA interference targeting methionine adenosyltransferase 2β gene (LV-shMAT2B) on hepatocelluar carcinoma (HCC) cells. METHODS: We constructed four plasmids of RNA interference targeting the MAT2B gene. After LV-shMAT2B was transfected with L-02 cells and two kinds of HCC cells, cell viability and proliferation were measured with MTT and [3H]thymidine assays respectively. Flow cytometry was used to assess cell apoptosis. The level of S-adenosyl methionine (SAMe) in HepG2 cells was evaluated. The expressions of cyclin D1, cyclin D2, bcl-xL and bcl-xS were detected with western blot. RESULTS: We constructed LV-shMAT2B successfully. LV-shMAT2B was safe for human normal liver cells. LV-shMAT2B caused dramatic reduction in proliferation compared with controls in HCC cells Bel-7402 (P = 0.054) and HepG2 (P = 0.031). Flow cytometry analysis showed that cell apoptosis caused by LV-shMAT2B was greater in HCC cells Bel-7402 and HepG2 than in control induced by scrambled siRNA (P = 0.047), but apoptosis rates in L-02 induced by LV-shMAT2B and scrambled siRNA respectively had no significant difference. Moreover, LV-shMAT2B significantly suppressed expression of MAT2B leading to growth-inhibition effect on HCC cells by down-regulating cyclin D1. Apoptosis induced by LV-shMAT2B was involved indown-regulating bcl-xL and up-regulating bcl-xS. CONCLUSION: LV-shMAT2B can induce cell apoptosis and growth-inhibition in HCC cells. MAT2B may be a therapy target in HCC in the future.

Construction and identification of recombinant lentivirus-mediated gene transfer system for rat transducer of regulated CREB activity 1

Objective： To construct a recombinant lentivirus vector which carries SD rat transducer of regulated CREB activity-1（TORC1） gene and examine its ability to express the TORC 1 gene in vitro. Methods： The coding sequence of SD rat TORC 1 gene was amplified using PCR and cloned into pGC-FU vector. 293T cells were transfected using Lipofectamine 2000 and packaged for the recombinant lentivirus particles. When the cloned sequence was identified to be right, the recombinant lentivirus particles were amplified in a large quantity. The titer of virus was determined by real-time PCR and the level of TORC1 expression was examined by Western blot. Results： The recombinant lentivirus vector carrying TORC1 was constructed successfully and could express TORC1 at a high level in 293T cells in vitro, and the titer determined by real-time PCR was 2 × 10^8 TU/ml. Conclusion： The recombinant lentivirus vector could express TORCl gene at a high level, and was very helpful in the study of exploring the effect of TORC1 on spinal cord injury.

靶向人c-Cbl基因重组干扰慢病毒与过表达腺病毒载体的构建、鉴定以及病毒功效研究

目的:构建可调控c-Cbl基因表达的重组慢病毒与腺病毒并评估其功效。方法:应用基因重组技术,分别构建靶向人c-Cbl基因的干扰慢病毒和过表达腺病毒。采用定量PCR和免疫印迹法检测病毒感染后白血病细胞(HL60、THP1)c-Cbl基因表达与转录本的变化。结果:3个靶向人c-Cbl基因的重组干扰慢病毒载体经测序验证构建成功,包装的病毒滴度均大于1×10^(8)TU/ml,其中shRNA-2号慢病毒干扰效率最高,白血病细胞感染后c-Cbl基因的表达约下调95%,CBL蛋白的表达约下调60%;同时,靶向人c-Cbl基因的重组过表达腺病毒载体也经测序验证构建成功,包装的病毒滴度大于1×10^(9)TU/ml,细胞感染腺病毒后,c-Cbl基因表达可瞬时上调约10倍,CBL蛋白表达约上调1.5倍。结论:重组干扰慢病毒和过表达腺病毒均可高效感染白血病细胞,并能分别下调和上调c-Cbl基因与CBL蛋白的表达,为后续研究肿瘤细胞内c-Cbl基因功能打下前期基础。

MAFA-PDX1过表达慢病毒感染人脐带间充质干细胞向胰岛素分泌细胞的分化

背景:干细胞来源胰岛β细胞移植被认为是治疗1型糖尿病的有效手段。人脐带间充质干细胞是理想的细胞来源,但其向胰岛β细胞分化的效率不高。目的:研究MAFA、PDX1修饰促进人脐带间充质干细胞向胰岛素分泌细胞分化的潜能。方法:构建MAFA-PDX1过表达慢病毒载体,用细胞形态学、RT-qPCR法、双硫腙染色比较3种方案[方案A:单纯慢病毒组;方案B:先药物(尼克酰胺、β-巯基乙醇)诱导再加慢病毒组;方案C:慢病毒和药物诱导同时进行组]诱导人脐带间充质干细胞分化为胰岛素分泌细胞的效率和潜能。结果与结论:(1)细胞形态学改变:经3种方案诱导后细胞形态均发生了改变,方案B在诱导第11天细胞聚集生长最为明显,出现聚集生长的胰岛样细胞团;(2)RT-qPCR检测胰岛相关基因的表达差异:同一诱导时间点3种方案横向比较,在诱导第5天,方案C的MAFA和PDX1基因表达量最高,方案B的GCG基因表达量最高;在诱导第11天,方案B的MAFA、PDX1基因表达量以及INS和GLUT2基因表达量最高;(3)双硫腙染色鉴定锌离子:3种方案诱导第11天部分细胞被双硫腙染成棕红色,其中方案B中部分小岛状细胞被染成棕红色,颜色较深(阳性表达);(4)结果表明,MAFA和PDX1共过表达可促进人脐带间充质干细胞向胰岛素分泌细胞分化;MAFA-PDX1基因修饰联合药物诱导方案优于单纯基因修饰方案。