Objective To study the nerve repair effect of olanzapine on schizophrenia model rats through its effect on cyclic AMP response element binding protein（CREB）/brain-derived neurotrophic factor（BDNF）/receptor tyrosine kinase receptors B（TrkB）pathway. Methods Total 60 rats were divided into control group, model group, olanzapine low, middle and high dose group. The rats in the model group, olanzapine low, middle and high dose groups were injected intraperitoneally with MK-801［0.2 mg/(kg ·d)］, while the control injected with the same amount of normal saline. The low, middle and high dose olanzapine groups were perfused with olanzapine solution of 0.5 mg/(kg ·d),1.0 mg/(kg ·d) and 1.5mg/(kg ·d) respectively. The behavior of rats was scored according to ataxia and stereotyped behavior standards, cognitive function and learning ability were evaluated by Morris water maze test, serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were detected by ELISA method, hippocampal histopathology was observed under microscope, and apoptosis and expression of CREB/BDNF/TrkB pathway related proteins in hippocampus were detected. Results Compared with the control group, the ataxia, the score of stereotyped behavior, the expression of TNF-α, IL-6 and the rate of apoptosis in the model group increased significantly (P<0.01). Compared with the control group, the number of crossing the platform, the time of staying in the target quadrant and the relative expression of CREB, p-CREB, p-TrkB, TrkB and BDNF protein in the model group decreased significantly (P<0.01), and those in the low and middle dose olanzapine groups decreased significantly (P<0.05). Compared with the model group, the times of crossing the platform and the stay time in the target quadrant increased significantly in the low and middle dose olanzapine groups (P<0.05). In the model group and the low dose olanzapine group, the hippocampal cells were swollen obviously, the nucleus was broken and divided, pyknosis, and the tissue arrangement was disorderly, while the phenomenon of fragmentation and nuclear pyknosis was rarely seen in the middle and high dose olanzapine groups. Conclusion The nerve repair mechanism of olanzapine on schizophrenic model rats is related to improving cognitive impairment, protecting hippocampal neurons and activating the expression of CREB/BDNF/TrkB signal pathway in rats.
Objective To investigate the structural distribution features and mechanism of elastic fibers and collagen fibers in ventricular interstitium of aged rats. Methods Five young SD rats (24 weeks) and five old SD rats (104 weeks) were used，and their cardiac function was examined by echocardiography. Modified Weigert elastic fiber staining, immunohistochemistry, immunofluorescence and Western blotting techniques were used to detect the expression changes of type I and Ⅲ collagen fibers and their proteins, elastic fibers and their proteins, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 2 (TIMP-2), respectively. Results The type I and type Ⅲ collagen in the ventricular interstitium of aged rats was very sufficient and wrapped around the cardiomyocytes. Compared with the young rats, the content of collagen protein in the ventricular interstitium of the aged rats significantly increased (P<0.05). Elastic fibers in the ventricular interstitium of the aged rats were and widely distributed. Compared with the young rats, the number of elastic fibers and the level of elastin in the ventricular interstitium of the aged rats significantly decreased (P<0.05), and the expression levels of MMP-2 and MMP-9 in ventricular muscle of aged rats increased, and they were correlated with the level of elastin. The level of TIMP-2 in ventricular muscle of aged rats decreased with age. Conclusion The number of collagen fibers and elastic fibers in ventricular interstitium of aged rats is fluctuated with each other. With the increase of age, the contents of TIMP-2 and elastic fibers in the ventricular interstitium gradually decreased, and the ratio of collagen fibers to elastic fibers is out of balance.
An improved fixation method for preparing mouse brown adipose tissue for transmission electron microscopy
Objective To improve the fixation method of the transmission electron microscope for better morphological preservation of mitochondria and lipid droplets in mouse brown adipose tissue. Methods The fixation method for mouse brown adipose tissue was optimized, mainly including an increased concentration of paraformaldehyde from 2% to 4% in the pre-fixative, employment of transcardial perfusion followed by immersion fixation in pre-fixation, and using imidazole-buffered osmium tetroxide as the post-fixative. The ultrastructures of brown adipocytes prepared by the improved method were observed and compared with those of a known standard protocol (3 mice in each group). The improved method was further validated in the quantitative analysis of mitochondrial cristae density and lipid droplets. Results The mitochondrial cristae and membrane structure of other organelles of brown adipocytes were better preserved using the optimized method compared with those of the standard method. Lipid droplets were presented as round structures with high electron density instead of vacuolated appearances. Using this method, we observed that the density of mitochondrial cristae and the content of lipid droplets increased in brown adipocytes after cold adaptation. Conclusion The optimized method can better preserve the ultrastructure of organelles in brown adipocytes, especially mitochondria and lipid droplets, and may be applicable for studying the ultrastructures remodeling of brown adipose tissue under different physiological or pathological conditions.
Objective To study the role of complement C3a receptor (C3aR) in cognitive impairment in rats with sepsis induced by cecal ligation and puncture (CLP), and to explore the possible mechanism. Methods Totally 132 rats were randomly divided into sham operation group (sham group) and sepsis group (CLP group). The initial number of each group was 66 animals, and 22 animals were set at each time point. The SD rat CLP animal model was constructed, and serum and brain (hippocampus and prefrontal cortex) samples were collected at day 1, day 15 and day 30 after operation. The levels of tumor necrosis factor-α(TNF-α), interleukin-1β (IL-1β) and IL-6 in the samples were determined by ELISA. Western blotting detected Tau protein (Tau), phosphorylated Tau（p-Tau）and C3aR expression in brain samples. Totally 66 rats were randomly divided into 3 groups, sham operation group, CLP group and C3aR antagonist(C3aRa) intervention group. On 15th, 17th, and 19th days after CLP, C3aRa intervened in rats, and they received inhibition avoidance test and object recognition test 30 days after CLP. The expressions of C3aR, lonized calcium binding adapter molecule 1(Iba1), GFAP and p-Tau in the hippocampus were detected by immunofluorescence. Results Compared with the sham group, the serum and brain tissue (TNFα, IL-1β and IL-6) of rats in the CLP group first increased and then decreased within 30 days after CLP. In the hippocampus and prefrontal cortex of CLP group, Tau phosphorylation was significantly enhanced at day 30 and day 1 and 15 after surgery, respectively (P<0.05). Compared with the sham group, C3aR protein levels in hippocampus and prefrontal cortex of rats in CLP group increased significantly at day 15 and 30 after operation (P<0.05). Compared with the CLP group, the endogenous C3aR content decreased significantly after C3aRa intervention (P<0.05), and Iba1, GFAP and p-Tau immunostaining were significantly inhibited (P<0.05). The C3aRa intervention in CLP rats reversed the cognitive impairment. Conclusion C3aR plays an important role in the development of biochemical and behavioral changes commonly associated with the onset of sepsis-induced neurodegenerative processes. C3aRa can be injected into the hippocampus to counteract the neurodegenerative process induced by sepsis.
Association between posterior tibial slope and tibial torsion angle and recurrent patellar dislocation based on the full-length CT of the lower limbs
Objective To measure and compare the lateral posterior tibial slope (LPTS), medial posterior tibial slope (MPTS) and tibial torsion angle (TTA) between the patients of recurrent patellar dislocation and the heathy people, and to analyze the correlation between LPTS, MPTS and TTA and the risk factors of recurrent patellar dislocation. Methods A total of 33 patients (44 knees) with recurrent patellar dislocation in our hospital from July 2019 to June 2021 were selected and listed as the study group. Twenty-three subjects (46 knees) who were suspected iliac vascular and lower limb vascular diseases during the same period were selected and listed as the control group. All the enrolled researchers had full-length CT scans date of the lower limbs. Three-dimensional models were reconstructed using Mimics 21.0 software and then imported into 3-matic software. The LPTS, MPTS and TTA were measured and compared between the two groups. Results In the study group, the LPTS, MPTS and TTA were （7.69±1.42）°,（10.06±1.71）°,（36.42±8.13）°, respectively, while the control group, the LPTS, MPTS and TTA were（8.42±1.65）°, （10.44±0.86）°, (25.77±3.90)°, respectively. There were no significant differences in the LPTS, MPTS and TTA between different genders and sides both in the study group and the control group (P>0.05). Compared with the control group, the LPTS in the study group was smaller, and the difference was statistically significant (P<0.05). There was no statistically significant difference between the study group and the control group in the MPTS (P>0.05). Compared with the control group, the TTA in the study group was higher, and the difference was statistically significant (P<0.05). Compared with the control group, the LPTS and MPTS in the study group were significant asymmetry, and the difference was statistically significant (P<0.05). Conclusion The lateral posterior tibial slope of patients with recurrent patellar dislocation is significantly smaller than that in the healthy people, while there is no significant difference in the medial posterior tibial slope; The tibial torsion angle of patients with recurrent patellar dislocation is significantly larger than in the healthy people; The lateral posterior tibial slope and tibial torsion angle have certain correlation with recurrent patellar dislocation, which can conduct the diagnosis of recurrent patellar dislocation.
Effect of interleukin-6 on nucleated erythrocytes in lipopolysaccharide induced preeclampsia rats
Objective To explore the effect of interleukin (IL)-6 on nucleated erythrocytes in lipopolysaccharide (LPS)-induced preeclampsia rats. Methods ELISA and immunohistochemistry were used to detect the IL-6 in peripheral blood and placenta of preeclampsia and normal pregnancy; Flow cytometry and immunofluorescence were used to detect the maternal nucleated erythrocytes. Pregnant SD rats were randomly divided into 3 groups: the control, LPS and LPS +anti-IL-6 group; IL-6, the proportion of nucleated erythrocytes, JAK2/MEK and PI3K/Akt signal-related genes were detected. Results The IL-6 of preeclampsia was higher than that of normal patients. Compared with the Control group, IL-6, the proportion of nucleated erythrocytes and JAK2, P85, Akt, P65, IL-1B mRNA of LPS group increased, the fetal weight decreased; Compared with the LPS group, IL-6, the proportion of nucleated erythrocytes and JAK2, P85, Akt, P65 and IL-1B mRNA of the LPS+anti-IL-6 group decreased. Conclusion The up-regulation of IL-6 of preeclampsia patients is accompanied by increased nucleated erythrocytes in peripheral blood. Neutralizing IL-6 in vivo may down-regulate JAK2/PI3K/Akt/NF-κB-signal-mediated IL-1B to protect preeclampsia rats.
Role of inhibiting lncRNA TUG1 to down-regulate nucleotide binding oligomerization domain like receptor protein 1 inflammasome in delaying the progression of Alzheimer’s disease
Objective To investigate the relieving effects of knockdown of long non-coding RNA（lncRNA）taurine up-regulated gene 1 (TUG1) on inhibiting nucleotide binding oligomerization domain like receptor protein 1(NLRP1) inflammasome and the progression of Alzheimer’s disease. Methods Wild-type (WT group,10 mice) or amyloid precursor protein (APP)/presenilin-1 (PS1) transgenic mice (30 mice) with a genetic background of C57/BL6 aged 9-10 weeks were used in this study. APP/PS1 transgenic mice were randomly divided into model group, model+lncRNA TUG1 short hairpin RNA (shRNA) group and model+shRNA non target（NT）group (n=10). Blood samples, cerebral cortex tissues, primary microglial cells and primary astrocytes were collected from mice 12 weeks of age on day 1 (3-month-old) and 32 weeks of age on day 1 (8-month-old), with 5 mice per group at each time point. Real-time PCR analysis was used to detect the expression levels of lncRNA TUG1 and macrophage migration inhibitory factor (MIF) mRNA in cerebral cortex tissues and primary microglial cells, and C1r and C1s mRNA levels in primary astrocytes of 3-month-old and 8-month-old mice in the above 4 groups, respectively. ELISA was used to determine the MIF in plasma samples of the above 4 groups of mice. Primary microglia and astrocytes from the cerebral cortex of 3-month-old and 8-month-old mice were co-cultured. CCK-8 method was used to determine the proliferation ability of the above cells. Western blotting was used to determine the expression levels of MIF, pro interleukin-1β(pro-IL-1β), apoptosis associated speck-like protein containing a caspase recrult domain(ASC), Caspase-1 (p20), Caspase-1 (full), NLRP1 and NLRP3 in cerebral cortex tissues of 3-month-old and 8-month-old mice. Immunofluorescent staining was used to determine amyloid beta（Aβ）in cerebral cortex of 8-month-old mice. Results At the age of 3-month-old and 8-month-old, compared with the WT group, the relative expression level of lncRNA TUG1 and MIF in cerebral cortex tissues and primary microglia of model group mice was significantly up-regulated, with primary microglial cells and astrocytes proliferation ability enhanced (P<0.05). Compared with the model group, the relative expression level of lncRNA TUG1 and MIF cerebral cortex tissues and primary microglia of model+lncRNA TUG1 shRNA group were significantly down-regulated, with primary microglial cells and astrocytes proliferation ability decreased (P<0.05). Compared with the WT group, MIF factor in the peripheral plasma of model group increased significantly, with pro-IL-1β，ASC，Caspase-1 (p20)，Caspase-1 (full), NLRP1 and NLRP3 expression level up-regulated in the model group mice cerebral cortex tissues, with increased Aβ immunofluorescent indensity (P<0.05). Compared with the model group, MIF factor in the peripheral plasma, and pro-IL-1β，ASC，Caspase-1 (p20)，Caspase-1 (full) and NLRP1 expression in the model+lncRNA TUG1 shRNA group mice cerebral cortex tissues were down-regulated, and Aβ immunofluorescent indensity decreased (P<0.05), while NLRP3 expression level were not changed (P>0.05). There was no significant difference between the model group and the model+shRNA NT group mice of all the above factors (P>0.05). Conclusion In APP/PS1 transgenic mice, up-regulation of lncRNA TUG1 and MIF are positively associated with the activation of NLRP1 inflammasome in mice cerebral cortex tissues and primary microglia. Knock-down of lncRNA TUG1 can ameliorate the progression of Alzheimer’s disease.
Objective The previous experiment showed that Ghrelin intervened in myocardial infarction leading to heart failure(HF) in rats at the early stage. The aim of this paper is to further investigate the correlation between growth differentiation facto-15 (GDF-15) and HF degree, which might lay a theoretical foundation of HF clinical treatments. Methods Eighty Wistar rats survived 6 hours in the modeling were randomly divided into sham operation(group A)( n =18), HF model (group B)( n =27), HF model of intervention(group C)( n =27). The blood GDF-15 expression level in 3 days，1 week，2 weeks，4 weeks and 8 weeks after surgery and the GDF-15 mRNA expression level in HF myocardial tissue in 2 weeks, 4 weeks and 8 weeks were tested by ELISA and real-time PCR. The rat myocardial ultrastructure was observed by electron microscope. Cardiac function was checked by B ultrasonic before executing the rats. Results Group B blood GDF-15 and myocardial tissue GDF-15 mRNA expression level were significantly higher than group C, but lower than group A. The difference was statistically significant( P <0.001).Ultrasonic testing results showed that group B heart increased significantly,left ventricular systolic and diastolic inner diameter increased obviously than group A and C in the 8 weeks. The difference was statistically significant ( P <0.001). Group B end-diastolic ventricular septal thickness, left ventricular thickness and left ventricular ejection fraction were significantly reduced than group A and C; In addition, myocardial ultrastructure shows group C change significantly than group B. Conclusion Ghrelin early intervention in HF in rats can improve heart function and reduce mortality; GDF-15 expression level can reflect HF lesions degree, and can be a new marker as auxiliary detection of HF.
Objective To observe the temporal and spatial changes of RUNX1T1 mRNA and protein expression in rat hippocampus after fimbria/fornix transection at different time points. Methods Rat models were prepared by cutting fimbria fornix, and divided into normal group, cutting 3days, 7days, 14days, 21days and 28days groups. 1. The total hippocampal tissue mRNA was extracted, and the expression of RUNX1T1 mRNA was detected by real-time PCR; 2. The total protein of hippocampus was extracted, and the expression of RUNX1T1 protein was detected by western blotting; 3. Frozen brain sections were detected by RUNX1T1 immunofluorescence and Hoechst labeled, to observe the RUNX1T1/Hoechst double labeled positive cells in hippocampal dentate gyrus. Results Real-time PCR analysis showed that the RUNX1T1 mRNA expression in the hippocampus was significantly upregulated on the 3rd day after transection. Western blotting analysis indicated that the expression of RUNX1T1 protein was detected in the hippocampus of rats at basal condation, and the levels of RUNX1T1 protein were markedly increased at days 3 and 7, and peaked at day 3 after transection. In contrast, no siginificant change in the RUNX1T1 protein expression at days 14, 21 and 28. Immunostaining showed that the nember of RUNX1T1-positive cells was siginificantly higher at day 3 compared with control and decreased slowly after day 7. Conclusion The transient upregulation of RUNX1T1 mRNA and protein expression in the early phase after fimbria/fornix transection, and location in the hippocampal dentate gyrus granule lower, which may be related to the hippocampal neural regeneration.
Objective To investigate the regulation mechanism of resveratrol(RES) on key enzymes of homocysteine metabolism for SD rats with insulin resistance. Methods Six weeks healthy mice were made as the insulin resistance model with diet by high fat and sugar and gavage by high fat for 3 months. The rats were randomly divided into the insulin resistance group (n=10)and the resveratrol intervention group (n=10). High fat and sugar were fed in the insulin resistance group for eight weeks. The resveratrol intervention group was fed by high fat and sugar and gavage by resveratrol［30mg/（kg·d)］ for eight weeks. The concentrations of homocysteine, glucose, insulin, total cholesterol and triglyceride were measured in the fasting state. The insulin resistance index (IRI) was calculated. The expressions of methylenetetrahydrofolate reductase (MTHFR), cystathionine β synthase (CBS) and methionine synthetase (MTR) were detected by immunohistochemical, RT-PCR and Western blotting in the rat liver tissue. Results The concentrations of the blood glucose, blood insulin and triglyceride in the resveratrol intervention group were lower than that in the insulin resistance group (P<0.05). The ISI was lower in the resveratrol intervention group. The concentration of homocysteine was decreased (P<0.05). However, the weight and the concentration of total cholesterol were no statistical significance (P>0.05). The protein expressions of MTHFR and CBS in the resveratrol intervention group were higher compared with the insulin resistance group by Western blotting and immunohistochemical staining(P<0.05).The mRNA expressions of MTHER and CBS in the resveratrol intervention group were higher compared with the insulin resistance group by RT-PCR. However, the expression of MTR protein and mRNA were not different between the resveratrol intervention group and the insulin resistance group (P>0.05). Conclusion Resveratrol can improve the expression of the MTHFR and CBS which participate the metabolism of homocysteine and facilitate the transformation of homocysteine, thus it may play a key role in alleviating insulin resistance.
Objective To explore the morphology, location and signal of the hypothalamic nuclei in the whole brain magnetic resonance imaging by comparing between magnetic resonance imaging（MRI）and sectional anatomy images of the same specimen of the human adult brain. Methods Six human adult cadaveric heads were examined using MRI and sectional anatomy methods. They were sectioned at horizontal, sagittal and coronal plane，two for each. The collected images from MRI and physical sections of the same brain were prepared. Results In T1 and T2 images, fornix and anterior commissure showed a sharp structure with hypo-signals,of which T1 images were even better. By a landmark such as the fornix or anterior commissure, the paraventricular nucleus in the T1 image showed sharp hypersignals, and slightly sharp hypersignals in T2 images. The mammillary nucleus showed a slightly sharp hypersignals in T1 images, and the mixed signals in the T2 images and the boundary was unclear. The supraoptic nucleus in the T1 image was less clear in boundaries with slightly hypersignals，in T2 images, it was poorly showed. Conclusion Hypothalamic nuclei can be identified in the routine MRI head scan.
Objective To investigate the effect of microRNA (miR)-138 regulation of Wnt signaling pathway on the biological behavior of human glioma cells in vitro. Methods Glioma cell lines U-87MG and U251 were selected and randomly divided into blank group, miR-NC group, miR-138 mimics group and miR-138 inhibitor group. Real-time PCR was used to detect the miR-138 expression in each group; MTT, flow cytometry, Transwell assay and scratch assay were used to detect proliferation, apoptosis, invasion and migration ability of each group respectively, and Western blotting was used to detect Wnt pathway related protein expression in each group. Results The miR-138 expression level was higher in the miR-138 mimics group compared with the remaining 3 groups, and that in the miR-138 inhibitor group was lower than that in the blank group and the miR-NC group (P<0.05); Compared with the blank group, the cell proliferation rate was lower in the miR-138 mimics group and higher in the miR-138 inhibitor group, and was time-dependent(P<0.05); The apoptosis rate in the miR-138 mimics group was higher than that in the blank group, miR-NC group, and miR-138 inhibitor group, while the apoptosis rate in the miR-138 inhibitor group was lower than that in the rest other groups(P<0.05); The number of cell-invading cells in the miR-138 mimics group was lower than that in the blank group, miR-NC group, and miR-138 inhibitor group, while all miR-138 inhibitor group were higher than the remaining three groups (P<0.05); The cell migration rate of miR-138 mimics group was lower than that of blank group, miR-NC group and miR-138 inhibitor group, while all miR-138 inhibitor group were higher than the remaining three groups (P<0.05); Wnt3a, Wnt1, glycogen synthase kinase 3β(GSK-3β) and β-catenin protein expression in the miR-138 mimics group was lower than that in the blank group, miR-NC group, and miR-138 inhibitor group; While miR-138 inhibitor groups were higher than the remaining three groups(P<0.05). Conclusion MiR-138 overexpression effectively inhibite the proliferation, invasion and migration of glioma cells and promote their apoptosis, probably achieved by pathway inhibition of the Wnt signaling pathway.
Objective To investigate the effect of sirtuin(SIRT)6/nuclear factor(NF)-κB signaling pathway in delaying hematopoietic stem cell (HSC) and progenitor cell(HPC) senescence with ginsenoside Rg1 during serial transplantation. Methods Sca-1+HSC/HPC from male donator mouse was isolated and purified by magnetic activated cell sorting (MACS). Sca-1+HSC/HPC replicability aged modelin vivo was established through the Sca-1+HSC/HPC serial transplantation. The femal recipient mice radiated lethal dose from 60Co γ ray were divided into four groups: the control group, the aged model group, the Rg1 treat aged group and the Rg1 prevent aged group. The effect of Rg1 to delay Sca-1+HSC/HPC senencence in vivo was evaluated by senescence-associated β-galactosidase (SA-β-gal) staining, mixed hematopoietic progenitor cell culture and cell cycle assay. The expressions of SIRT6 and NF-κB mRNA and protein were detected by quantitative PCR and Western blotting. Results With the increasing of transplantation, Sca-1+HSC/HPC appeared aging characters. The number of cells entered G0/G1 phase and the percentage of SA-β-gal positive cells was increased, and the number of CFU-Mix was decreased. Compared with the aged model group, the number of cells entered G0/G1 phase and the percentage of SA-β-gal positive cells were decreased, the number of CFU-Mix was increased to Sca-1+ HSC/HPC in Rg1 treat aged group and Rg1 prevent aged group, the expression of SIRT6 mRNA and protein was up regulated and the expression of NF-κB mRNA and protein was down regulated in Rg1 treat aged group and Rg1 prevent aged group.The changes of Rg1 prevent aged group was significantly higher than Rg1 treat aged group. Conclusion Rg1 could resist Sca-1+HSC/HPC senescence during serial transplantation in which the signaling pathway of SIRT6-NF-κB may play an important role.
Objective Tibetan teenagers in Tibet by counting their muscle mass with bioelectrical impedance method. Methods We selected randomly 1427 healthy teenagers whose parents were Tibetan in Tibet (710 males, 717 females).They had signed the informed consent.We detected all subjects with the body composition analyzer and concluded the total muscle, trunk(left upper limb,right upper limb,left lower limb,right lower limb)muscle mass.All the results were inputted SPSS(statistical software) and processed by independent sample t-test and variance analysis. Results The total muscle mass, trunk(left upper limb,right upper limb,left lower limb,right lower limb)muscle mass of males were all more than those of females in all age groups of Tibetan teenagers( P <0.05 or P <0.01).In general,the muscle mass of Tibetan teenagers in Tibet increased with age,males’ increasing trend was more pronounced. Conclusion The muscle development of Tibetan teenagers in Tibet is related with age and hormone secretion,it reflects the physiological characteristics in different developmental stages.
Objective To establish a cell bank of mouse induced pluripotent stem cells (iPSCs), which would be a useful resource for the research of stem cells and iPSCs. Methods C57BL/6J mouse embryonic fibroblasts were transfected with lentivirus with Sox2, Oct4, Klf4 and c-Myc. Twelve days after viral infection, mouse iPSCs clones were picked and expanded, the alkaline phosphatase (AP) was stained. The iPSCs were hanging cultured in vitro and subcutaneously inoculated into BABL/c nude mice. All-trans retinoic acid (RA) was introduced to the media to induce smooth muscle cell (SMC) differentiation of iPSCs, and SMC-specific gene (smMHC and SMA) expression were measured by RT-PCR analyses. PCR method was employed for species identification and detection of mycoplasma. Results Twelve AP positive mouse iPSCs clonal strains were picked and expanded, and 9 of them formed teratomas in BALB/c nude mice. HE staining of the teratoma showed various types of tissues. RT-PCR of iPSCs in RA media showed positive expression of smMHC and SMA. The iPSCs clonal strains were identified as murine species, without mycoplasma contamination was found, and were deposited by cell resource center. Conclusion A model of mouse iPSCs was successfully established, which may provide available resources for reprogramming and directed differentiation mechanism research of ES cells and iPSCs.
Objective To investigate the expression of interleukin（IL)-34/ colony stimulating factor（CSF）-1R in the process of transforming growth factor（TGF）-β1 inducing epithelial-mesenchymal transition (EMT) of human alveolar epithelial cells A549 cells. Methods A549 cells were cultured in vitro. CCK 8 was used to test the influence of the proliferative rate of A549 cells which were stimulated by TGF-β1 at different concentrations and time points. A549 cells were stimulated by 5μg/L TGF-β1 at 0 hour, the 12th hour, the 24th hour, and the 48th hour. Western blotting was adopted to detect changes of the following proteins: α-smooth muscle actin(α-SMA), E-cadherin（E-Cad）, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1(TIMP-1). Real-time PCR was adopted to detect changes of the following genes: IL-34 mRNA, CSF-1R mRNA, MMP-2 mRNA and MMP-9 mRNA. Results TGF-β1 had no significant influence in the proliferation of A549 cells compared with the control group(P>0.05). TGF-β1（5μg/L）stimulated A549 cells at different time point (0 hour, 12, 24, 48 hours), compared with the control group. The epithelial phenotype E-Cad protein was gradually down-regulated(P<0.01), while the mesenchymal phenotype α-SMA protein was gradually up-regulated(P<0.01) and the protein of MMP-2 increased gradually (P<0.01). The protein of MMP-9 increased firstly and then was reduced(P<0.01),the peak was at the 24th hour. The protein of TIMP-1 was firstly transiently increased and then reduced(P<0.01)， the minimum was at the 48th hour. Compared with the control group, the gene of IL-34 mRNA increased gradually (P<0.01), and the genes of CSF-1R mRNA, MMP-2 mRNA and MMP-9 mRNA increased firstly and then decreased(P<0.01)， which were peaked respectively at the 24th hour, the 24th hour, the 12th hour, respectively. Conclusion In the process of TGF-β1 inducing A549 cells transition，there is accompanied with the expression of IL-34/CSF-1R.
