ABSTRACT
Objective:To establish a fluorescent reporter human induced pluripotent stem cell line (hiPSCs) for monitoring the expression of visual system homeobox 2 ( VSX2). Methods:VSX2_small guide RNA (sgRNA) was inserted into vector PX459 to construct knockout plasmid, and the P2A-eGFP knock-in donor plasmid was conducted at the same time.The two plasmids were transfected into BC1-hiPSCs.Single cell clones were generated after treatment of puromycin.Correct insertion was confirmed by PCR and Sanger sequencing.The isogenicity of the parental and the reporter hiPSCs was confirmed by STR analysis and karyotyping.Pluripotency capacity of the reporter hiPSCs was analysed by reverse trascription PCR and immunofluorescence.Three-germ-layer formation experiment was carried out to analyse the multi-lineage differentiation ability of the reporter hiPSCs.The reporter hiPSCs were further differentiated to obtain three-dimension (3D) retinal organoids, and immunofluorescence was used to identify the co-localization of the enhanced green fluorescent protein (eGFP) and VSX2.Results:A VSX2 eGFP reporter hiPSC clone was successfully obtained by CRISPR/Cas9 technology, which was consistent with the parental hiPSCs (BC1-hiPSCs) in morphology, without any chromosomal aberrations or cell line cross-contamination.Reverse transcription PCR assay and immunofluorescence showed obvious positive expressions of iPSCs markers in BC1- VSX2 eGFP-iPSCs, including NANOG, OCT4, SOX2, DNMT3B and GDF3 mRNA as well as NANOG, OCT4, SSEA4 and TRA-1-60 protein.The α-fetoprotein (AFP), α-smooth muscle actin (α-SMA) and neuronal class Ⅲ β-tubulin (TUJ1) were expressed in endoderm, mesoderm and ectoderm, respeetively, derived from BC1- VSX2 eGFP-iPSCs, and eGFP and VSX2 were co-stained in the neural retinal layer of 3D retinal organoids derived from BC1- VSX2 eGFP-iPSCs by immunofluorescence. Conclusions:VSX2 fluorescent reporter hiPSCs is successfully generated, which can monitor the temporal and spatial expression changes of VSX2 protein in real time, providing a powerful tool for evaluation of retina development mechanism and cell therapy.
ABSTRACT
Objective:To evaluate the retinal differentiation ability of human induced pluripotent stem cells (hiPSCs) from various somatic cell sources.Methods:The hiPSCs lines BC1- green fluorescent protein (GFP) and Gibco obtained by blood cell reprogramming and the hiPSCs line UE017 obtained by urine cell reprogramming were used to induce retinal differentiation.The morphogenesis and development of retina were recorded with an optical microscope, and the expression of specific molecular markers of various cell subclasses in the retina was detected by immunofluorescence, and the efficiency of retinal differentiation of different cell lines was analyzed and compared.Results:All three hiPSC lines derived from blood and urine cells were able to be induced into three-dimensional (3D) retinal organoids, including neuroretina and retinal pigment epithelial cells.Retinal organoids simulated the development process of retina in vivo and gradually differentiated into all cell subtypes of retina, including retinal ganglion cells, photoreceptor cells, amacrine cells, horizontal cells, bipolar cells, Müller cells, and even formed lamellar structures.However, in terms of the efficiency of acquiring retinal organoids, the hiPSCs derived from blood were more efficient than those derived from urine. Conclusions:hiPSCs from both blood and urine somatic cells can differentiate into 3D retinal organoids, including all subtypes of retinal cells.The differentiation efficiency among lines is different.
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Objective To observe the effect of sinomenine (SIN) on the expression of cyclooxygenase (COX2),alpha-7 nicotinic acetylcholine receptor(α7nAChR) and adenosine receptor(A2A) in A549 cells,and to explore the relative mechanism for cell proliferation.Methods The effect of SIN and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on the proliferation of A549 cells was determined by methyl thiazolyl tetrazolium (MTT) assay.The effect of SIN and NNK on the migration of A549 cells was detected by cell wound scratch assay.The effect of SIN and NNK on COX2 expression in A549 cells was determined by Western blotting method.The effect of SIN and NNK on the expression of α7nAChR and A2A mRNA and protein was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting method.Results NNK increased the proliferation and migration of A549 cells,while SIN inhibited the proliferation and migration of A549 cells.COX2 expression level was increased in NNK group but was decreased in SIN group.The expression levels of α7nAChR and A2A were up-regulated in NNK group but were down-regulated in SIN group.Conclusion SIN plays a role in inhibiting the proliferation and migration of A549 cells by suppressing COX2 expression.SIN has an inhibitory effect on the expression of α7nAChR and A2A.
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Objective To explore the effect of β-element on the oadiosensitivity of transplanted tumor, and its relationship with the expression of survivin. Methods The transplanted mice model was established through the cell suspension inoculation. The mice with transplanted tumor size of 0. 8-1.0 cm3 were randomly divided into 8 groups as blank control, 25, 45 and 100 mg/kg group, irradiation group,25 mg/kg + irradiation group, 45 mg/kg + irradiation group, 100 mg/kg + irradiation group. The tumor size was measured every other day until tumor size was double, and the growth curve was obtained. The average tumor growth inhibition rate of β-element and tumor size were attained at 2,4,6 and 8 d after β-element injection. The expression of survivin was detected with immunohistochemistry. Results The nude mice model was successfully established and the growth curves were obtained according to the tumor size.Between 2 and 8 d after β-elemene injection, the variation tendency of the average tumor growth inhibition rate was consistent with the size in β-elemene treatment groups. The antitumor effect of β-elemene was in a dose-dependent manner. The values of radiosensitivity enhancement factor were 0. 84,1.24,2.04 for 25,45 and 100 mg/kg group, respectively ,and the optimal dose was 45 mg/kg. β-element had little effect on the expression of survivin, and the expression of survivin significantly enhanced in irradiation group and decreased in β-element + irradiation groups. Conclusions β-elemene could enhance the tumor radiosensitivity through inhibitiong the expression of survivin.