This study was conducted to investigate the paclitaxel loaded by hydrazone bonds in poly(ethylene glycol)-poly(caprolactone) micelles (mPEG-PCL-PTX) on proliferation and apoptosis of human lung cancer A549 cells and its possible mechanisms of anti-tumor activity. The cell proliferation was measured with MTT assay. Flow cytometry were used to analyze the cell cycle. The cell apoptosis was analyzed using Hoechst/P staining. The expression levels of apoptotic genes expression in the mitochondrial apoptosis pathway were detected by RT-PCR and Western blotting, respectively. The mPEG-PCL-PTX could inhibit the proliferation of A549 cells and promote the apoptosis. The Bax, caspase-3 protein expression were increased while Bcl-2 protein expression was decreased in A549 cells. Results showed that the polymer containing hydrazone bond is non-toxic in vitro, the mPEG-PCL-PTX micelles can inhibit the proliferation and induce the apoptosis of A549 cells. Key words: paclitaxel; micelle; A549 cell; proliferation; cell cycle; apoptosis
Apoptosis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Micelles ; Paclitaxel ; pharmacology ; Polyesters ; Polyethylene Glycols ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo study the changes of microRNA expression in cortex tissues in neonatal rats with hypoxic-ischemic brain damage (HIBD)and the possible roles of microRNA in the pathogenesis of HIBD. METHODS Rat HIBD model was prepared. The cortex tissues were obtained 14 days after the HIBD event. The microRNA expression profiles were measured using microRNA microarray. Expression of 9 microRNAs (miR-126,-26a,-674-5p,-21,-25,-290, miR-124,-125b-5p and microRNA-9a) was determined by quantitative real-time PCR.

RESULTShe results of microRNA expression profiles indicated that 27 pieces of microRNA were up-regulated more than 2 folds and 60 pieces were down-regulated more than 2 folds compared with the normal control group. The results of the 9 microRNAs detected by quantitative real-time PCR were consistent with those detected by microRNA microarray.

CONCLUSIONSHIBD rats have significant changes in microRNA expression, suggesting that microRNA expression may play important roles in the pathogenesis of HIBD.

Animals ; Animals, Newborn ; Apoptosis ; Cell Cycle ; Hypoxia-Ischemia, Brain ; etiology ; genetics ; MicroRNAs ; genetics ; physiology ; Oligonucleotide Array Sequence Analysis ; Polymerase Chain Reaction ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of p65 gene inhibited by siRNA on neuronic differentiation in the marrow mesenchymal stem cells (MSCs).

METHODSThe MSCs were transfected with Rn-p65-siRNA. Fasudil hydrochloride induced MSCs differentiating into neurons. The non-transfected group and negative control group (transfected with negative control siRNA marked by Cy3) were used as controls. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope at 24 h,48 h and 72 h after transfected with negative control siRNA. The viability of MSCs was detected by MTT at 24 h, 48 h and 72 h after transfected with Rn-p65-siRNA. The expressions of p65 mRNA and protein in MSCs were detected by RT-PCR and Western blot respectively. The expressions of p65 protein, NSE, MAP-2 and glial fibrillary acidic protein (GFAP) were detected by immunocytochemical method after transfection for 6 h.

RESULTSThe fluorescence of MSCs was mostly displayed after transfection of 72 hours and the efficiency of transfection was up to 83.3% ± 3.8%. Meanwhile, the p65 mRNA and p65 protein expressed by MSCs of transfected group were significantly decreased (P < 0.05); MTT displayed that the viability of MSCs was also significantly reduced (P < 0.05). The best efficiency of induction was observed in the transfected group. There were higher expressions of NSE and MAP-2 than the other group (P < 0.05).

CONCLUSIONThe p65 gene inhibited by siRNA can promote the marrow mesenchymal stem cells to differentiate into neurons.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; Animals ; Cell Differentiation ; Glial Fibrillary Acidic Protein ; metabolism ; Mesenchymal Stromal Cells ; cytology ; Neurons ; cytology ; RNA, Messenger ; RNA, Small Interfering ; Rats ; Transcription Factor RelA ; antagonists & inhibitors ; metabolism ; Transfection

OBJECTIVETo study the effects of Down syndrome cellular adhesion molecule (DSCAM) on differentiation of rat marrow mesenchymal stem cells (MSCs) into neurons in vitro.

METHODSMSCs from Sprague-Dawley rats were induced into neurons by baicalin. The expression of DSCAM before and after induction was evaluated by immunocytochemical staining and Western blot assay. After knockdown of DSCAM by siRNA transfection, the differentiation rate of neurons derived from MSCs was measured.

RESULTSBefore induction, the expression of DSCAM was not detectable in MSCs. After bFGF preinduction for 24 hrs, DSCAM was slightly expressed in MSCs (1.71+/- 0.67%). The DSCAM expression increased 6 hrs after baicalin induction (15.79+/- 4.24%), reached a peak at 3 days (53.16+/- 5.94%) and then decreased gradually. The DSCAM expression 6 days after baicalin induction (28.99+/- 6.72%) was significantly lower than that at 3 days (P<0.01). However, after DSCAM-siRNA transfection, the DSCAM expression in MSCs was significantly reduced. MSCs did not express neuron-specific beta-III-tubulin before induction. After baicalin induction for 6 hrs, 3 days and 6 days, the expression of beta-III-tubulin was 1.40+/- 0.79%, 41.59+/- 3.17% and 59.11+/- 4.76% respectively. But the beta-III-tubulin expression significantly decreased 3 and 6 days after DSCAM-siRNA transfection (28.57+/- 2.91% and 43.90+/- 12.31% respectively).

CONCLUSIONSDSCAM may play an important role in MSCs differentiation into neural cells.

Animals ; Bone Marrow Cells ; cytology ; Cell Adhesion Molecules ; physiology ; Cell Differentiation ; Mesenchymal Stromal Cells ; cytology ; Neurons ; cytology ; RNA, Small Interfering ; genetics ; Rats ; Rats, Sprague-Dawley ; Transfection

To establish SP2/0 cell line H-2(d) stably expressing bcr-abl fusion gene fragment, the bcr-abl fusion gene was subcloned into retroviral vector pLXSN from pGEMbcr-abl. The recombinant retroviral vector pLXSNbcr-abl was transfected into PT67 packaging cells with the help of lipofectamine. The positive clones were selected out and cultured after G418 selection. Then viral supernatant was collected to determine viral titer, the viral titer was 2 x 10(7) CFU/ml. The SP2/0 cells were infected with the collected viral supernatant. The results showed that after G418 selection, the bcr-abl fusion gene was integrated into the chromosome of SP2/0 cells infected stably, with recombinant retrovirus and expressed in SP2/0 cells confirmed by PCR and RT-PCR respectively. In conclusion, the mouse tumor cell lines expressing the bcr-abl fusion protein were successfully established and would be used as a experimental cell model for anti-CML immunotherapy.
Animals ; Cell Line ; Cell Line, Tumor ; Fusion Proteins, bcr-abl ; genetics ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; genetics ; Humans ; K562 Cells ; Mice ; Mice, Inbred BALB C ; Multiple Myeloma ; genetics ; pathology ; NIH 3T3 Cells ; Peptide Fragments ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Retroviridae ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; methods

OBJECTIVETo evaluate the effects of glial cell line-derived neurotrophic factor (GDNF) and memantine on the long-term prognosis in neonatal rats with ischemia-induced periventricular leukomalacia (PVL).

METHODSThirty-two 5-day-old neonatal rats were randomly divided into 4 groups: sham-operated, PVL, GDNF-treated and memantine-treated. PVL was induced by right carotid artery ligation and hypoxia in the PVL, GDNF-treated and memantine-treated groups. GDNF (100 μg/kg) or memantine (20 mg/kg) was injected in the two treatment groups immediately after PVL inducement. The weight of the rats was measured immediately before and after hypoxia ischemia (HI). Both of Morris water maze test and Rivlin inclined plane test were performed at 26 days old (21 days after HI). The values of the escape latency (EL) and swimming distance, and the maximum inclined plane degree which the rats could stand at least 5 seconds were compared among the four groups.

RESULTSThe lower weight, the prolonged mean values of EL and swimming distance and the reduced maximum inclined plane degree were observed in the PVL group compared to those in the sham-operated, GDNF-treated and memantine-treated groups. There were no significant differences in the weight, the values of EI and swimming distance and the maximum inclined plane degree between the two treatment groups and the sham-operated group.

CONCLUSIONSThe administration of either GDNF or memantine can markedly increase the abilities of spatial discrimination,learning and memory, and motor coordination, promote weight gain, and improve long-term prognosis in rats with PVL.

Animals ; Animals, Newborn ; Body Weight ; Excitatory Amino Acid Antagonists ; therapeutic use ; Glial Cell Line-Derived Neurotrophic Factor ; therapeutic use ; Humans ; Infant, Newborn ; Leukomalacia, Periventricular ; drug therapy ; psychology ; Maze Learning ; drug effects ; Memantine ; therapeutic use ; Motor Activity ; drug effects ; Rats

Autosomal recessive cerebellar ataxias (ARCA) are a highly heterogeneous group of rare neurodegenerative diseases affecting both central and peripheral nervous systems. Based on pathological mechanisms, five major types of ARCA may be distinguished, which include mitochondrial ataxia, metabolic disorder, DNA repair defect ataxia, congenital ataxias and degenerative ataxia. This review summarizes clinical features, molecular genetics and recent advances in DNA sequencing of common types of ARCA.
Cerebellar Ataxia ; classification ; genetics ; metabolism ; Genes, Recessive ; Humans

OBJECTIVETo investigate the influence of mIL-7 on the immune response induced by vaccine of bcr-abl fusion gene fragment in mouse.

METHODSBALB/c mice were immunized by i. m. injection of pVbcr-abl/mIL-7 and pVbcr-abl, respectively. The specific antibody to p210bcr-abl protein was assayed by ELISA. The CTL activity of spleen cells from the immunized mice was assessed with LDH release test.

RESULTSThe pVbcr-abl/mIL-7 and pVbcr-abl-immunized BALB/c mice elicited higher specific antibodies to p210bcr-abl protein. The specific antibody level of former group was higher than that in latter group, but the difference was statistically not significant. The spleen cells from the immunized mice showed more effective CTL activity than that from control group. The cytotoxic activity of spleen CTLs induced by pVbcr-abl/mIL-7 immunized mice exceeded that of pVbcr-ab-immunized mice.

CONCLUSIONThe mIL-7 may influence the growth and differentiation of T cells, promote some T cells migrating into tumor tissue and up-regulate the specific cellular immune response. The results of this study provided an useful experimental basis for preclinical research on gene vaccine for chronic myeloid leukemia.

Animals ; Antibodies ; blood ; Cancer Vaccines ; genetics ; immunology ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Fusion Proteins, bcr-abl ; biosynthesis ; genetics ; immunology ; Humans ; Interleukin-7 ; biosynthesis ; genetics ; immunology ; K562 Cells ; Mice ; Mice, Inbred BALB C ; Random Allocation ; Spleen ; cytology ; T-Lymphocytes, Cytotoxic ; immunology ; Vaccination ; Vaccines, DNA ; immunology

Objective To investigate the impacts of LINC00943 on proliferation,apoptosis,and invasion of esophageal squamous cell carcinoma(ESCC)cells by regulating the miR-126-5p/HOXB2 axis.Methods QRT-PCR was applied to detect the expression of LINC00943 in 45 ESCC tissues and cell lines;The effects of LINC00943 knockdown on the proliferation,apoptosis and invasion of KYSE30 cells were detected by MTT assay,flow cytometry and Transwell chamber.Western blot was applied to detect the expression of proliferating cell nuclear antigen(PCNA),Bcl-2 associated X protein(Bax),anti apoptotic factor B cell lymphomatoma-2(Bcl-2),cleaved caspase-3,matrix metalloproteinase-2(MMP-2),MMP-9,and HOXB2;dual luciferase reporter gene experiment was applied to verify the relationship between miR-126-5p and LINC00943 and HOXB2;the ESCC nude mouse in vivo model was constructed and separated into si-NC and si-LINC00943 groups,the tumor mass and volume were measured,qRT-PCR was applied to detect the expression of miR-126-5p in transplanted tumor tissue,immunohistochemistry was applied to detect the expression of HOXB2 protein in transplanted tumor tissue,and TUNEL staining was applied to detect cell apoptosis in tumor tissue.Results The expression of LINC00943 mRNA increased in ESCC tissues and cell lines(P＜0.05);Compared with the si-NC group,the proliferation activity,migration and invasion cell number,HOXB2,PCNA,MMP-2,MMP-9 and Bcl-2 protein expression of KYSE30 cells in the si-LINC00943 group were decreased,and the expression of miR-126-5 p,Bax,Cleaved Caspase-3 and apoptosis rate were increased(P＜0.05);downregulation of miR-126-5p was able to weaken the inhibitory effect of LINC00943 knockdown on the malignant behavior of KYSE30 cells(P＜0.05);dual luciferase reporter gene experiment confirmed the targeted regulatory relationship between miR-126-5p and LINC00943,and miR-126-5p and HOXB2(P＜0.05);The ESCC nude mouse model was constructed in vivo and divided into si-NC and si-LINC00943 groups.The tumor mass,tumor volume,expression of miR-126-5p and HOXB2 in transplanted tumor tissues and apoptosis rate of tumor cells were measured.(P＜0.05).Conclusion LINC00943 is highly expressed in ESCC tissues and cell lines.Knocking down LINC00943 may inhibit the expression of HOXB2 by up-regulating the expression of miR-126-5p,promote the apoptosis of ESCC cells,and inhibit their proliferation,migration and invasion.

OBJECTIVETo investigate the effect of notch signaling on differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neurons induced by fasudil hydrochloride.

METHODSThe experiments were divided into non-transfected group, transfected group (transfected with Rn-Notch1-siRNA), positive control group (transfected with Rn-MAPK-1 Control siRNA) and negative control group (transfected with negative control siRNA). Fasudil hydrochloride induced MSCs differentiating into neurons. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope. The expression of notch1 mRNA, Hes1 mRNA and MAPK1 mRNA in MSCs was detected by RT-PCR. The expression of Notch1 protein, NSE, neurofilament M (NF-M) and glial fibrillary acidic protein(GFAP)was detected by immunocytochemical method. The viability of MSCs was detected by MTT.

RESULTS(1) The fluorescence of MSCs was mostly displayed after transfection for 72 h and the efficiency of transfection was up to 91.3% +/- 4.2%. Meanwhile, the notch1 mRNA and Hes1 mRNA expressed by MSCs of transfected group were significantly decreased (P < 0.05) and MTT displayed that the viability of MSCs was also significantly reduced (P < 0.05). (2) Fasudil hydrochloride could induce MSCs differentiate into neurons and the best efficiency of induction was observed in the transfected group. There was higher expression of NSE and neurofilament-M (NF-M) than the other groups (P < 0.05).

CONCLUSIONThere may be notch1 signaling and Rho/Rho GTPase signaling synergy on differentiation of rat bone marrow stromal cell into neurons induced by fasudil hydrochloride and they jointly promote the differentiation of MSCs into neurons.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; analogs & derivatives ; pharmacology ; Animals ; Bone Marrow Cells ; cytology ; drug effects ; Cell Differentiation ; drug effects ; Cells, Cultured ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Neurons ; cytology ; Rats ; Rats, Wistar ; Receptor, Notch1 ; metabolism ; Signal Transduction