OBJECTIVE: To explore the interaction between Tubulin beta 4B class IVb (TUBB4B) and Agtpbp1/cytosolic carboxypeptidase- like1 (CCP1) in mouse primary spermatocytes (GC-2 cells) and the role of TUBB4B in regulating the development of GC-2 cells. METHODS: Lentiviral vectors were used to infect GC-2 cells to construct TUBB4B knockdown and negative control (NC-KD) cells. The stable cell lines with TUBB4B overexpression (Tubb4b-OE) and the negative control (NC-OE) cells were screened using purinomycin. RT-qPCR and Western blotting were used to verify successful cell modeling and explore the relationship between TUBB4B and CCP1 expressions in GC-2 cells. The effects of TUBB4B silencing and overexpression on the proliferation and cell cycle of GC-2 cells were evaluated using CCK8 assay and flow cytometry. The signaling pathway proteins showing significant changes in response to TUBB4B silencing or overexpression were identified using Western blotting and immunofluorescence assay and then labeled for verification at the cellular level. RESULTS: Both TUBB4B silencing and overexpression in GC-2 cells caused consistent changes in the mRNA and protein expressions of CCP1 (P < 0.05). Similarly, TUBB4B expression also showed consistent changes at the mRNA and protein after CCP1 knockdown and restoration (P < 0.05). TUBB4B knockdown and overexpression had no significant effect on proliferation rate or cell cycle of GC-2 cells, but caused significant changes in the key proteins of the nuclear factor kappa-B (NF-κB) signaling pathway (p65 and p-p65) and the mitogen-activated protein kinase (MAPK) signaling pathway (ErK1/2 and p-Erk1/2) (P < 0.05); CCP1 knockdown induced significant changes in PolyE expression in GC-2 cells (P < 0.05). CONCLUSIONS TUBB4B and CCP1 interact via a mutual positive regulation mechanism in GC-2 cells. CCP-1 can deglutamize TUBB4B, and the latter is involved in the regulation of NF-κB and MAPK signaling pathways in primary spermatocytes.
Animals ; Male ; Mice ; GTP-Binding Proteins/metabolism* ; Mitogen-Activated Protein Kinases/metabolism* ; NF-kappa B/metabolism* ; RNA, Messenger ; Serine-Type D-Ala-D-Ala Carboxypeptidase/metabolism* ; Signal Transduction ; Spermatocytes ; Tubulin/genetics*

OBJECTIVE: To demonstrate the mechanism of mechanical ventilation (MV) induced endoplasmic reticulum stress (ERS) promoting mechanical ventilation-induced pulmonary fibrosis (MVPF), and to clarify the role of angiotensin receptor 1 (AT1R) during the process. METHODS: The C57BL/6 mice were randomly divided into four groups: Sham group, MV group, AT1R-shRNA group and MV+AT1R-shRNA group, with 6 mice in each group. The MV group and MV+AT1R-shRNA group mechanically ventilated for 2 hours after endotracheal intubation to establish MVPF animal model (parameter settings: respiratory rate 70 times/minutes, tidal volume 20 mL/kg, inhated oxygen concentration 0.21). The Sham group and AT1R-shRNA group only underwent intubation after anesthesia and maintained spontaneous breathing. AT1R-shRNA group and MV+AT1R-shRNA group were airway injected with the adeno-associated virus one month before modeling to inhibit AT1R gene expression in lung tissue. The expressions of AT1R, ERS signature proteins [immunoglobulin heavy chain-binding protein (BIP), protein disulfide isomerase (PDI)], fibrosis signature proteins [collagen I (COL1A1), α-smooth muscle actin (α-SMA)] in lung tissues were detected by immunofluorescence and Western blotting. Hematoxylin-eosin (HE) staining was used to evaluate lung injury and Masson staining was used to evaluate pulmonary fibrosis. RESULTS: Compared with the Sham group, the degree of pulmonary fibrosis and lung injury were more significant in the MV group. In the MV group, the protein expressions of AT1R, BIP, PDI, COL1A1 and α-SMA were increased (AT1R/β-actin: 1.40±0.02 vs. 1, BIP/β-actin: 2.79±0.07 vs. 1, PDI/β-actin: 2.07±0.02 vs. 1, COL1A1/α-Tubulin: 2.60±0.15 vs. 1, α-SMA/α-Tubulin: 2.80±0.25 vs. 1, all P < 0.01). The number of E-cad⁺/AT1R⁺ and E-cad⁺/BIP⁺ cells in lung tissue increased, and the fluorescence intensity of COL1A1 and α-SMA increased. Compared with the MV group, the degree of pulmonary fibrosis and lung injury were significantly relieved in the MV+AT1R-shRNA group. In the MV+AT1R-shRNA group, the protein expressions of AT1R, BIP, PDI, COL1A1 and α-SMA were decreased (AT1R/β-actin: 0.53±0.03 vs. 1.40±0.02, BIP/β-actin: 1.73±0.15 vs. 2.79±0.07, PDI/β-actin: 1.04±0.07 vs. 2.07±0.02, COL1A1/α-Tubulin: 1.29±0.11 vs. 2.60±0.15, α-SMA/α-Tubulin: 1.27±0.10 vs. 2.80±0.25, all P < 0.01). The number of E-cad⁺/AT1R⁺ and E-cad⁺/BIP⁺ cells in lung tissue decreased, and the fluorescence intensity of COL1A1 and α-SMA decreased. There was no statistically significant difference in the indicators between AT1R-shRNA group and Sham group. CONCLUSIONS MV up-regulate the expression of AT1R in alveolar epithelial cells, activate the AT1R pathway, induce ERS and promote the progression of MVPF.
Mice ; Animals ; Pulmonary Fibrosis/chemically induced* ; Lung Injury ; Respiration, Artificial/adverse effects* ; Actins/metabolism* ; Tubulin ; Mice, Inbred C57BL ; Endoplasmic Reticulum Stress ; RNA, Small Interfering

Objective: To investigate the regulatory effects of bio-intensity electric field on directional migration and microtubule acetylation in human epidermal cell line HaCaT, aiming to provide molecular theoretical basis for the clinical treatment of wound repair. Methods: The experimental research methods were used. HaCaT cells were collected and divided into simulated electric field group (n=54) placed in the electric field device without electricity for 3 h and electric field treatment group (n=52) treated with 200 mV/mm electric field for 3 h (the same treatment methods below). The cell movement direction was observed in the living cell workstation and the movement velocity, trajectory velocity, and direction of cosθ of cell movement within 3 h of treatment were calculated. HaCaT cells were divided into simulated electric field group and electric field treatment 1 h group, electric field treatment 2 h group, and electric field treatment 3 h group which were treated with 200 mV/mm electric field for corresponding time. HaCaT cells were divided into simulated electric field group and 100 mV/mm electric field group, 200 mV/mm electric field group, and 300 mV/mm electric field group treated with electric field of corresponding intensities for 3 h. The protein expression of acetylated α-tubulin was detected by Western blotting (n=3). HaCaT cells were divided into simulated electric field group and electric field treatment group, and the protein expression of acetylated α-tubulin was detected and located by immunofluorescence method (n=3). Data were statistically analyzed with Kruskal-Wallis H test,Mann-Whitney U test, Bonferroni correction, one-way analysis of variance, least significant difference test, and independent sample t test. Results: Within 3 h of treatment, compared with that in simulated electric field group, the cells in electric field treatment group had obvious tendency to move directionally, the movement velocity and trajectory velocity were increased significantly (with Z values of -8.53 and -2.05, respectively, P<0.05 or P<0.01), and the directionality was significantly enhanced (Z=-8.65, P<0.01). Compared with (0.80±0.14) in simulated electric field group, the protein expressions of acetylated α-tubulin in electric field treatment 1 h group (1.50±0.08) and electric field treatment 2 h group (1.89±0.06) were not changed obviously (P>0.05), while the protein expression of acetylated α-tubulin of cells in electric field treatment 3 h group (3.37±0.36) was increased significantly (Z=-3.06, P<0.05). After treatment for 3 h, the protein expressions of acetylated α-tubulin of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 300 mV/mm electric field group were 1.63±0.05, 2.24±0.08, and 2.00±0.13, respectively, which were significantly more than 0.95±0.27 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of acetylated α-tubulin in 200 mV/mm electric field group and 300 mV/mm electric field group were increased significantly (P<0.01); the protein expression of acetylated α-tubulin of cells in 300 mV/mm electric field group was significantly lower than that in 200 mV/mm electric field group (P<0.05). After treatment for 3 h, compared with that in simulated electric field group, the acetylated α-tubulin of cells had enhanced directional distribution and higher protein expression (t=5.78, P<0.01). Conclusions: Bio-intensity electric field can induce the directional migration of HaCaT cells and obviously up-regulate the level of α-ubulin acetylation after treatment at 200 mV/mm bio-intensity electric field for 3 h.
Humans ; Acetylation ; Tubulin/metabolism* ; Microtubules/metabolism* ; Electricity ; Epidermal Cells/metabolism*

Objective: To observe the intestinal time-dependent changes in Parkinson's disease (PD) mouse model constructed by intraperitoneal injection of paraquat (PQ) and to establish the brain-gut axis connection initially. Methods: In October 2019, 48 mice were randomly divided into treated group and control groups: treated 4-week (P-4) group, treated 6-week (P-6) group, treated 8-week (P-8) group, control 4-week (C-4) group, control 6-week (C-6) group, and control 8-week (C-8) group. The treated group was injected with 15 mg/kg PQ solution and the control group was injected with 0.9% saline (0.2 ml/20 g) by intraperitoneal injection twice a week. After the initial state (0 weeks) and the treatment at the end of 4, 6 and 8 weeks, the mood changes and motor functions of mice were assessed by neurobehavioral tests (open field test, pole climbing test, tail suspension test and elevated plus maze test) . And the number of fecal pellets for 1 h and water content were calculated to assess the functional status of the gastrointestinal tract. Western blotting experiments were performed to detect the expression levels of α-synuclein (α-syn) and tyrosine hydroxylase (TH) in the nigrostriatal region of the mouse brain, the tight junction markers zonula occludens-1 (ZO-1) and Occludin, the inflammatory markers of integrin αM subunit (CD11b) , inducible nitric oxide synthase (iNOS) , high mobility group box 1 (HMGB1) , interleukin-1β (IL-1β) , and the neuronal markers βⅢ-tubulin and α-syn protein in the colon.Immunohistochemical staining was performed to detect the expression levels of colonic tight junction proteins ZO-1 and Occludin. Immunofluorescence staining was performed to detect the expression levels of TH in the substantia nigra region of the midbrain, and the co-localization of colonic intestine neuronal marker (βⅢ-tubulin) and Ser129 α-syn in the colonic. Results: Compared with the initial state (0 weeks) and C-8 group, mice in the P-8 group had significantly higher pole climbing test scores and resting time, and significantly lower total active distance, mean active speed, percentage of open arm entry and 1 h fecal instances (P<0.05) . After poisoning, the 1 h fecal water content of model mice first increased and then decreased, the P-4 and P-6 groups were significantly higher than the simultaneous point control group, and the P-8 groups were significantly lower than the initial state (P<0.05) . Compared with control, P-4 and P-6 groups, the expression levels of ZO-1 and Occludin in the P-8 group were significantly decreased (P<0.05) . Compared with control group, the expression levels of CD11b and IL-1β in the P-4 group were significantly increased (P<0.05) . Compared with control and P-4 group, the expression levels of CD11b, iNOS, HMGB1 and IL-1β in the P-6 and P-8 groups were significantly increased (P<0.05) . Compared with the control and P-4 groups, the expression levels of βⅢ-tubulin in the colon of mice in the P-8 group were significantly decreased, and the expression levels of α-syn and Ser129 α-syn were significantly increased (P<0.05) . The expression level of Ser129 α-syn in the colon of model mice was negatively correlated with the expression level of βⅢ-tubulin (r(s)=-0.9149, 95%CI: -0.9771--0.7085, P<0.001) . Ser129 α-syn and βⅢ-tubulin co-localization in the colonic intermuscular plexus region increased gradually with the time of exposure. Compared with the control, P-4 and P-6 groups, the expression level of TH in the nigrostriatal region of the brain was significantly decreased, and the expression levels of α-syn and Ser129 α-syn were significantly increased in the P-8 group (P<0.05) . Correlation analysis showed that the relative expression level of Ser129 α-syn in the nigrostriatal region of the brain was negatively correlated with the expression level of TH in the model mice (r(s)=-0.9716, 95% CI: -0.9925--0.8953, P<0.001) . Conclusion: The PD mouse model is successfully established by PQ, and the intestinal function of the model mice is reduced in a time-dependent manner. And on this basis, it is preliminary determined that the abnormal aggregation of α-syn may be an important substance connecting the brain-gut axis.
Animals ; Brain-Gut Axis ; Disease Models, Animal ; HMGB1 Protein ; Intestines ; Mice ; Mice, Inbred C57BL ; Occludin ; Paraquat/toxicity* ; Parkinson Disease ; Tubulin ; Tyrosine 3-Monooxygenase/metabolism* ; Water

OBJECTIVE: To investigate the the effects of leptin on the proliferation, differentiation and PTEN expression of rat retinal progenitor cells (RPCs) cultured under hypoxic condition. METHODS: SD rat RPCs were cultured in normoxic conditions or exposed to hypoxia in the presence of 0, 0.3, 1.0, 3.0, 10, and 30 nmol/L leptin for 12, 48 and 72 h, and the cell viability was assessed using cell counting kit 8 (CCK 8) assay. The RPCs in primary culture were divided into control group, hypoxia group, and hypoxia+leptin group, and after 48 h of culture, the cell medium was replaced with differentiation medium and the cells were further cultured for 6 days. Immunofluorescence staining was employed to detect the cells positive for β-tubulin III and GFAP, and Western blotting was used to examine the expression of PTEN at 48 h of cell culture. RESULTS: The first generation of RPCs showed suspended growth in the medium with abundant and bright cellular plasma and formed mulberry like cell spheres after 2 days of culture. Treatment with low-dose leptin (below 3.0 nmol/L) for 48 h obviously improved the viability of RPCs cultured in hypoxia, while at high concentrations (above 10 nmol/L), leptin significantly suppressed the cell viability (P < 0.05). The cells treated with 3.0 nmol/L leptin for 48 h showed the highest viability (P < 0.05). After treatment with 3.0 nmol/L leptin for 48 h, the cells with hypoxic exposure showed similar GFAP and β-tubulin Ⅲ positivity with the control cells (P>0.05), but exhibited an obvious down-regulation of PTEN protein expression compared with the control cells (P < 0.05). CONCLUSION In rat RPCs with hypoxic exposure, treatment with low dose leptin can promote the cell proliferation and suppress cellular PTEN protein expression without causing significant effects on cell differentiation.
Animals ; Cell Differentiation/drug effects* ; Cell Hypoxia/drug effects* ; Cell Proliferation/drug effects* ; Cells, Cultured ; Leptin/pharmacology* ; PTEN Phosphohydrolase/metabolism* ; Rats ; Rats, Sprague-Dawley ; Retina/metabolism* ; Stem Cells/metabolism* ; Tubulin

OBJECTIVE: To investigate the mechanism by which doublecortin promotes the recovery of cytoskeleton in arginine vasopressin (AVP) neurons in rats with electrical lesions of the pituitary stalk (PEL). METHODS: Thirty-two SD rats were randomized into PEL group with electrical lesions of the pituitary stalk through the floor of the skull base (=25) and sham operation group (=7), and the daily water consumption (DWC), daily urine volume (DUV) and urine specific gravity (USG) of the rats were recorded. Four rats on day 1 and 7 rats on each of days 3, 7 and 14 after PEL as well as the sham-operated rats were sacrificed for detection of the expressions of β-Tubulin (Tuj1), doublecortin and caspase- 3 in the AVP neurons of the supraoptic nucleus using immunofluorescence assay and Western blotting. RESULTS: After PEL, the rats exhibited a typical triphasic pattern of diabetes insipidus, with the postoperative days 1-2 as the phase one, days 3-5 as the phase two, and days 6-14 as the phase three. Immunofluorescent results indicated the repair of the AVP neurons evidenced by significantly increased doublecortin expressions in the AVP neurons following PEL; similarly, the expression of Tuj1 also increased progressively after PEL, reaching the peak level on day 7 after PEL. The apoptotic rates of the AVP neurons exhibited a reverse pattern of variation, peaking on postoperative day 3 followed by progressive reduction till day 14. Western blotting showed that the expressions of c-Jun and p-c-Jun were up-regulated significantly on day 3 ( < 0.05) and 7 ( < 0.01) after PEL, while an upregulated p-JNK expression was detected only on day 3 ( < 0.05), as was consistent with the time-courses of neuronal recovery and apoptosis after PEL. CONCLUSIONS JNK/c-Jun pathway is activated after PEL to induce apoptosis of AVP neurons in the acute phase and to promote the repair of neuronal cytoskeleton by up-regulation of doublecortin and Tuj1 expressions.
Animals ; Apoptosis ; Arginine Vasopressin ; pharmacology ; Cytoskeleton ; metabolism ; MAP Kinase Signaling System ; Neurons ; cytology ; Pituitary Gland ; cytology ; injuries ; Proto-Oncogene Proteins c-jun ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Regeneration ; Tubulin ; metabolism

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Animals ; Brain/pathology ; *Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry/pharmacology ; Dental Pulp/*cytology ; Dopaminergic Neurons/*cytology/*metabolism/pathology ; Enzyme-Linked Immunosorbent Assay ; Glial Fibrillary Acidic Protein/genetics/metabolism ; Humans ; Mice ; Mice, Inbred ICR ; Myelin Basic Protein/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Stage-Specific Embryonic Antigens/genetics/metabolism ; Stem Cells/*cytology/*metabolism/pathology ; Tubulin/genetics/metabolism ; Tyrosine 3-Monooxygenase/analysis/genetics/metabolism

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Animals ; Brain/pathology ; *Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry/pharmacology ; Dental Pulp/*cytology ; Dopaminergic Neurons/*cytology/*metabolism/pathology ; Enzyme-Linked Immunosorbent Assay ; Glial Fibrillary Acidic Protein/genetics/metabolism ; Humans ; Mice ; Mice, Inbred ICR ; Myelin Basic Protein/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Stage-Specific Embryonic Antigens/genetics/metabolism ; Stem Cells/*cytology/*metabolism/pathology ; Tubulin/genetics/metabolism ; Tyrosine 3-Monooxygenase/analysis/genetics/metabolism

To explore whether hypoxic condition could promote the olfactory mucosa mesenchymal stem cells (OM-MSCs) to differentiate into neurons with the olfactory ensheathing cells (OECs) supernatant and the potential mechanisms.
 Methods: The OM-MSCs and OECs were isolated and cultured, and they were identified by flow cytometry and immunofluorescence. The OM-MSCs were divided into three groups: a 3%O2+ HIF-1α inhibitors (lificiguat: YC-1) + OECs supernatant group (Group A) , a 3%O2 + OECs supernatant group (Group B) and a 21%O2 + OECs supernatant group (Control group). The neurons, which were differentiated from OM-MSCs, were assessed by immunofluorescence test. The mRNA and protein expression of hypoxia-inducible factor-1α (HIF-1α), βIII-tubulin and glial fibrillary acidic portein (GFAP) were detected by quantitative polymerase chain reaction (Q-PCR) and Western blot. The potassium channels were analyzed by patch clamp.
 Results: The neurons differentiated from OM-MSCs expressed the most amount of βIII-tubulin, and the result of Q-PCR showed that HIF-1α expression in the Group B was significantly higher than that in the other groups (all P<0.05). Western blot result showed that the βIII-tubulin protein expression was significantly higher and GFAP protein expression was obviously decreased in the Group B (both P<0.05). The patch clamp test confirmed that the potassium channels in the neurons were activated.
 Conclusion: Hypoxic condition can significantly increase the neuronal differentiation of OM-MSCs by the OECs supernatant and decrease the production of neuroglia cells, which is associated with the activation of HIF-1 signal pathway.
Blotting, Western ; Cell Differentiation ; physiology ; Cells, Cultured ; Culture Media, Conditioned ; chemistry ; pharmacology ; Flow Cytometry ; Glial Fibrillary Acidic Protein ; metabolism ; Hypoxia ; physiopathology ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Indazoles ; pharmacology ; Mesenchymal Stem Cells ; physiology ; Neurogenesis ; physiology ; Neuroglia ; metabolism ; physiology ; Neurons ; physiology ; Olfactory Mucosa ; Potassium Channels ; Signal Transduction ; Tubulin ; metabolism

Pigs are increasingly recognized as "natural" hosts of infection by human respiratory viruses because of their similarities to humans in terms of lung physiology, airway morphology, cell types, and distribution of cell receptors in the respiratory tract. We wished to explore the mechanisms of infection by respiratory viruses and screening of drug that could be used to treat respiratory-system diseases. Hence, we developed a model of well-differentiated porcine airway epithelial cells (PAECs) derived from pig-lung tissue and cultured them with serum-free medium under an air-liquid interface condition in vitro. We identified the PAEC model using scanning electron microscopy, electrophysiology, and immunohistology. To evaluate application of gene therapy of adeno-associated virus (AAV)6 on the PAEC model, we generated recombinant adeno-associated virus 6-green fluorescent protein (rAAV6-GFP) using the three-plasmid transfection method and infected PAECs from the apical surface with rAAV6-GFP. Results demonstrated that the PAEC model comprised a multilayer epithelial structure containing ciliated mucous secretory cells, with basal cells located directly beneath the multilayer. rAAV6-GFP could infect PAECs from the apical surface and efficiently transduce PAECs to mediate the long-term expression of the exogenous gene. Establishment of a model of well-differentiated PAECs in vitro could lay a solid foundation for the study of infection by respiratory pathogens, as well as the screening and gene therapy of agents used to treat diseases of the respiratory system.
Animals ; Cell Differentiation ; Dependovirus ; genetics ; Epithelial Cells ; cytology ; metabolism ; Green Fluorescent Proteins ; genetics ; HEK293 Cells ; Humans ; Lung ; cytology ; Membrane Potentials ; Mucins ; metabolism ; Swine ; Transduction, Genetic ; Tubulin ; metabolism